The effect of blood flow restriction techniques during aerobic exercise in healthy adults

Transcription

1 The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2015 The effect of blood flow restriction techniques during aerobic exercise in healthy adults Trent E. Cayot University of Toledo Follow this and additional works at: Recommended Citation Cayot, Trent E., "The effect of blood flow restriction techniques during aerobic exercise in healthy adults" (2015). Theses and Dissertations This Dissertation is brought to you for free and open access by The University of Toledo Digital Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of The University of Toledo Digital Repository. For more information, please see the repository's About page.

2 A Dissertation Entitled The Effect of Blood Flow Restriction Techniques during Aerobic Exercise in Healthy Adults By Trent E Cayot Submitted to the Graduate Faculty as partial fulfillment of the requirements for The Doctor of Philosophy Degree in Exercise Science Barry Scheuermann, Ph.D., Committee Chair Suzanne Wambold, Ph.D., RN, Committee Member Michael Tevald, Ph.D., P.T., Committee Member David Weldy, M.D., Ph.D., Committee Member Patricia Komuniecki, Ph.D., Dean College of Graduate Studies The University of Toledo May 2015

3

4 An Abstract of The Effect of Blood Flow Restriction Techniques during Aerobic Exercise in Healthy Adults by Trent E Cayot Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Exercise Science The University of Toledo May 2015 Although the importance of aerobic exercise in disease prevention and maintenance of a healthy lifestyle has been extensively demonstrated [1-4], it was recently reported by the American Heart Association (AHA) that approximately 30% of the adult population within the United States does not engage in regular aerobic exercise [2]. The most commonly reported reason why adults did not engage in regular exercise was due to a "lack of time" within their daily routine [5, 6]. In order to best integrate exercise into a time constrained schedule many have turned to high-intensity interval training (HIIT) due to the advantageous training outcomes reported in a relatively short duration (2-4 week) [7, 8]. In addition, the exercise volume is significantly reduced (~80-90%) during HIIT sessions compared to traditional "continuous" cardiovascular exercise sessions [8, 9] thus decreasing the time spent exercising [8]. However, the exercise intensities used during HIIT sessions ("all-out effort" [9, 10] or near maximal intensities [11, 12]) may become a deterrent or may not be appropriate for certain populations. An exercise technique known as blood flow restriction (BFR) exercise may be an acceptable alternative approach for these populations as it utilizes low exercise iii

5 intensities. BFR exercise has been shown to concurrently increase muscle hypertrophy [13, 14], muscle strength [13] and peak oxygen uptake (VO 2pk ) [14, 15] subsequent to low-intensity (i.e., walking, cycling) cardiovascular training programs. The combination of BFR (i.e., decreased exercise intensity) and interval training (i.e., decreased exercise volume) is both intriguing and a unique alternative solution that could potentially be applicable to a variety of populations. This alternative exercise approach (i.e., BFR interval training) addresses many commonly cited barriers for exercise retention (i.e., time constrained schedules, high exercise intensities). Therefore, the primary purpose of this dissertation was to determine the results of a short duration (2 weeks) BFR low-intensity interval training (BFR-LIIT) program on aerobic capacity and skeletal muscle strength (chapter 5). However, before the primary purpose could be investigated many secondary aims needed to be examined, including i) determining the effect of occlusion duration on the microvascular oxygenation and neuromuscular activation during exercise (chapter 3) and ii) determining the acute physiological responses (oxygen uptake, microvascular oxygenation, neuromuscular activation) to BFR used in cardiovascular exercise models (constant load, chapter 4; interval, chapter 5). The effects of occlusion duration were examined as healthy subjects performed isometric knee extension contractions at different sub-maximal intensities under control (CON, no occlusion), immediate occlusion (IO) and pre occlusion (PO) conditions. During the IO condition the occlusion pressure (130% of the resting systolic blood pressure, 130% SBP) was applied immediately prior to exercise while the occlusion pressure (130% SBP) was applied five minutes prior to exercise in the PO condition. iv

6 Varying the occlusion duration did not affect the neuromuscular activation of the exercising musculature (p > 0.05), although activation did significantly increase with increasing sub-maximal exercise intensities. However, PO elicited greater microvascular deoxygenation (deoxy-[hb+mb]), as assessed by near-infrared spectroscopy) compared to CON at all exercise intensities (p < 0.05), whereas the deoxy-[hb+mb] was only greater during PO compared to IO at the lowest exercise intensity tested (20% maximal voluntary contraction, MVC). Furthermore, IO resulted in greater deoxy-[hb+mb] compared to CON only at low exercise intensities (20% MVC, 40% MVC). In conclusion, although occlusion duration did significantly affect neuromuscular activation, BFR techniques influenced microvascular oxygenation the most during low-intensity exercise. Many investigations have observed an increased neuromuscular activation with BFR resistance exercise [16-19], however, the peripheral responses (i.e., neuromuscular activation, microvascular oxygenation) to BFR cardiovascular exercise (i.e., cycling) has yet to be determined. Therefore, healthy subjects performed bouts of heavy (above estimated lactate threshold, >LT) constant cycling exercise with and without BFR. No difference in oxygen uptake (VO 2 ) was observed (p > 0.05) despite a greater deoxy- [Hb+Mb] response during the beginning and end of BFR exercise compared to control (CON) exercise (p < 0.05). Unlike previous BFR resistance training investigations [16-19], BFR cycling exercise resulted in significantly lower neuromuscular activation during the end of exercise. Additionally each exercise condition elicited an increase in blood lactate concentration (from 20 watt baseline cycling to immediately post-exercise), however, plasma vascular endothelial growth factor receptor 2 was not significantly v

7 affected subsequent to any exercise condition. These results may suggest that the perturbation caused by BFR during low-intensity cycling exercise may have a greater localized affect within the exercising muscle, similar to previous investigations [20-23]. Lastly, healthy subjects completed a short duration BFR low-intensity interval training (BFR-LIIT) program on a cycle ergometer. The subjects performed 8-12 intervals at 40% VO 2pk during six exercise sessions across two weeks. During the BFR- LIIT sessions continuous bilateral occlusion was applied to the proximal thigh at an occlusion pressure of 130% SBP. Significant increases in the estimated LT and knee extensor strength (isometric, eccentric) were observed following BFR-LIIT. However, no changes were detected in VO 2pk and oxidative phosphorylation capacity at the level of the mitochondria (assessed from the phase II oxygen uptake time constant). Collectively all of the investigations suggest that the perturbation induced by BFR techniques during cardiovascular exercise has a greater localized affect within the exercising musculature. Furthermore, we suggest that exercise volume is more heavily relied upon to induce significant training stimuli during BFR exercise since the exercise intensity is reduced. This could explain the lack of increase in VO 2pk (3.3%) following BFR-LIIT as a low exercise volume (interval exercise, 2 weeks) was combined with lowintensity exercise. Therefore, the findings within this dissertation would not recommend the use of BFR during short duration (2 weeks), low volume (interval) exercise programs if the training objectives include significant peak cardiovascular adaptations (VO 2pk ). Future investigation into an appropriate dose response of BFR low-intensity exercise and exercise volume is required to explain previous reports of increases in VO 2pk subsequent to BFR training [14, 15]. However, rapid improvements in muscle strength and sub- vi

8 maximal aerobic capacity (estimated LT) were observed with BFR-LIIT that may have considerable applicability to certain populations. vii

9 Acknowledgements I would like to express my sincere gratitude and appreciation to Dr. Barry Scheuermann for all of his time, guidance and expertise during my undergraduate and graduate years at the University of Toledo. Dr Scheuermann although you've taught me many lessons during the past years, I will never forget the Black Swan Hypothesis and the importance of asking a well-developed question. I would also like to thank my dissertation committee members (Dr Suzanne Wambold, Dr Michael Tevald and Dr David Weldy) for their time, critical analysis, discussions of the present investigations. Mr. Jakob Lauver, I am extremely appreciative and grateful for the many hours spent discussing research at the white board, collaborative efforts and your friendship outside the research lab. I wish you nothing but the best with your future endeavors in a successful career in exercise physiology and I hope to continue our collaborations in the future. I would like to thank the wonderful members of my family and close friends for their continued support and encouragement during this process. Grandpa thank you for always being such a positive influence in my life and teaching me the value of work ethic. Lastly, I would like to thank my beautiful wife Marci for all of her patience, love and support. I truly would not have been able to have accomplished this without you. viii

10 Table of Contents Abstract... ii Acknowledgements... viii List of Tables... xi List of Figures... xi i 1 Introduction Literature Review Effects of Blood Flow Restriction Duration on Muscle Activation and Microvascular Oxygenation During Low-Volume Isometric Exercise Introduction Methods Results Discussion Conclusion Acute Effects of Blood Flow Restriction During Heavy Intensity Cycling Exercise Introduction Methods Results ix

11 4.4 Discussion Conclusion Effects of Blood Flow Restriction Low Intensity Interval Training on Cardiovascular Endurance and Maximal Strength in Healthy Adults Introduction Methods Results Discussion Conclusion Conclusion References Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Appendix G x

12 List of Tables 4.1 Subject Demographics Plasma VEGF-R2 Concentrations Plasma Lactate Concentrations Subject Demographics Training Adaptations...95 xi

13 List of Figures 3-1 Vastus Lateralis RMS vs. Intensity Vastus Medialis RMS vs. Intensity Microvascular Deoxygenation vs. Intensity Total Hemoglobin Concentration vs. Intensity Vastus Lateralis RMS vs. Exercise Duration Microvascular Deoxygenation vs. Exercise Duration Total Hemoglobin Concentration vs. Exercise Duration Oxygen Uptake vs. Exercise Duration Peak Oxygen Uptake Training Responses Estimated Lactate Threshold Training Responses Phase II Oxygen Uptake Time Constant Training Responses Peak Concentric Strength Training Responses Peak Eccentric Strength Training Responses Peak Isometric Strength Training Responses Oxygen Uptake vs. Interval Microvascular Deoxygenation vs. Interval Total Hemoglobin Concentration vs. Interval...91 xii

14 5-10 Average RMS vs. Interval Mean Rating of Perceived Exertion vs. Interval...93 xii

15 Chapter 1 Introduction Individuals depend on aerobic and anaerobic energy systems in order to perform increasing amounts of physical exertion (such as activities of daily living, recreational activities, occupational demands, sport, etc). The increasing amount of physical exertion to perform such activities necessitates an increase in oxygen uptake (VO 2 ) to match the energy requirement of the exercising muscles [24]. Peak voluntary oxygen uptake (VO 2pk ) has previously been shown to be an important cardiopulmonary measurement that is often used to classify fitness levels [1], prescribe exercise intensities [1] and to predict mortality risk [25]. Furthermore, VO 2pk has been previously demonstrated to be "trainable" as significant increases in VO 2pk (~10%) have occurred subsequent to a variety of cardiovascular exercise programs [10, 14, 26] and in addition has been associated with increases in peak work capacity (WR pk ) and time to exhaustion [27]. The American College of Sports Medicine (ACSM) recognizes this ability to train VO 2pk via cardiovascular exercise programs and therefore recommends that a healthy adult participate in 5-7 days of moderate intensive (40%-60% VO 2pk ) aerobic exercise or 3 days of vigorous intensive ( 60%VO 2pk ) aerobic exercise each week [1]. The health benefits of regular cardiovascular exercise, such as the previously mentioned ACSM 1

16 recommendations, include decreased risk for many chronic cardiovascular, pulmonary, and metabolic diseases [1-4]. Although research has demonstrated the importance of aerobic exercise in disease prevention [1-4], it was recently reported by the American Heart Association (AHA) that approximately 30% of the adult population within the United States does not engage in regular aerobic exercise [2]. The most common explanation provided by adults as to why they are not able to engage in regular exercise or physical activity is due to a "lack of time" within their daily routine [5, 6]. Also noted in the same report issued by the AHA was the observation that 18% of girls and 10% of boys in grades 9-12 reported leading a sedentary lifestyle by not engaging in physical activity at least one day per week [2]. This is an alarming statistic as the increased occurrence of a sedentary lifestyle that is often observed within the adult population is being portrayed within the youth population of the United States. A sedentary lifestyle has been defined as a modifiable risk factor for cardiovascular disease [1], however, approximately one-third of all deaths within the United States are due to cardiovascular disease [2]. These statistics are clear evidence that further scientific investigations are necessary to i) assist in integrating regular cardiovascular exercise into a time constrained daily routine and ii) further investigate the role of regular cardiovascular exercise in disease prevention strategies thereby developing effective exercise prescriptions for a wide variety of populations. An approach to exercise programming known as high-intensity interval training (HIIT) has gained a considerable amount of attention and has been promoted because of the decrease in exercise volume associated with most HIIT programs. Indeed, many HIIT programs have an exercise volume that is approximately 80-90% less than most 2

17 traditional continuous aerobic exercise programs [8, 9]. HIIT sessions include repeated intermittent short-duration, high-intensity bouts of exercise followed by short-duration, low-intensity exercise bouts [28]. The high-intensity exercise bouts during HIIT sessions are typically performed at exercise intensities equivalent to an "all-out effort" [9, 10] or at near maximal intensities [11, 12]. Rodas and colleagues demonstrated that a short-term HIIT program completed over a two week (14 HIIT sessions) training period using all-out exercise intensities significantly increased VO 2pk within a healthy, recreationally active male population [10]. Furthermore, muscle biopsies obtained from the vastus lateralis indicated significant increases in citrate synthase following the HIIT program [10]. Citrate synthase is the enzyme responsible for catalyzing acetyl-coa and oxaloacetate to form citrate in the first step of the tricarboxylic acid (TCA) cycle [29] and has been used extensively as a marker of muscle aerobic capacity, particularly following exercise training [30]. In agreement with Rodas and colleagues [10], VO 2pk and citrate synthase was shown to significantly increase following a HIIT program in a healthy, recreationally active, female population using near-maximal exercise intensities (90% VO 2pk ) and a lower exercise volume (13 days, 7 HIIT sessions) [12]. Little and colleagues [7] reported similar training adaptations as significant elevations in citrate synthase following a short-term, lowvolume HIIT program (2 weeks, 6 HIIT sessions, 100%VO 2pk ) within a healthy, recreational active male population. Citrate synthase responses subsequent to a HIIT program have been shown to be similar to the citrate synthase response following a traditional cardiovascular exercise program (65% VO 2pk, min per session, 5 days per week) despite the significant differences in exercise volumes [9]. Other important 3

18 biomarkers (peroxisome proliferator-activated receptor γ coactivator 1 alpha, PGC-1α) that promote mitochondrial biogenesis [31, 32] have been previously reported to be upregulated following the completion of HIIT programs [9, 33]. Although HIIT appears to be an attractive alternative exercise option due to the physiological improvements [27] and the reduction in exercise volume [8, 9], a portion of the exercise session is performed at relatively high-intensities ("all-out effort"[9, 10], near maximal [7, 11]). The high-intensity exercise could become a deterrent to an individual due to the relatively high amount of effort required or may not be suitable for some exercising populations. Therefore, this dissertation will investigate the implementation of a rather unique exercise technique known as blood flow restriction (BFR) exercise during the performance of aerobic exercise. Previous scientific investigations have used BFR techniques combine with lowintensity exercise in order to induce metabolic stress [34-36] and mimic high-intensity exercise in both resistance exercise models [37-43] and cardiovascular (cycling, walking) exercise models [13-15, 44-47]. VO 2pk has been previously shown to increase in healthy young men following both cycling and walking programs that incorporated BFR [14, 15]. Not only was an increase in VO 2pk observed following a BFR cycling program [14], but muscular endurance, measured as an increase in time to task failure, and muscular hypertrophy, as evidenced by an increase in muscle cross-sectional area, have also been shown to increase compared to a typical low-intensity (40% VO 2pk, 45 min) cycling exercise program [14]. In addition, previous BFR investigations utilizing low-intensity walking programs have demonstrated increases in anaerobic capacity, assessed by the 4

19 Wingate test [15], stroke volume [15], skeletal muscle hypertrophy [13, 44], skeletal muscle dynamic strength [13], and skeletal muscle isometric strength [13, 44]. One possible mechanistic explanation for the aerobic training adaptations following BFR aerobic exercise programs could be due to increases in vascular endothelial growth factor (VEGF) concentrations. VEGF has been established as a potent exercise-induced angiogenic stimulator [48, 49] and has been associated with the formation of new capillaries and improvements in oxygen delivery to exercising skeletal muscle [49-52]. The increased VEGF expression, previously observed subsequent to BFR resistance exercise [43, 53, 54], could promote angiogenesis during a BFR cardiovascular training program and potentially improve oxygen delivery and utilization and thus subsequently increase VO 2pk. However, to our knowledge the response of plasma VEGF has not been previously observed following acute BFR aerobic exercise (Chapter 4). In addition, many methodological considerations arise regarding BFR prior to implementing BFR techniques into an aerobic exercise program. Mainly, how does the occlusion duration affect the local muscular environment (Chapter 3)? This is an important consideration as the increases in metabolic stress and hypoxic intramuscular environments have been suggested as necessary exercise stimuli during the performance of BFR exercise [35, 36, 55]. Therefore, the primary objectives of this dissertation is to i) determine the peripheral effects of BFR implemented into a cardiovascular exercise model and ii) to determine the physiological training adaptations that result from BFR low-intensity cardiovascular training programs and then compare the training outcomes to those observed from a high-intensity cardiovascular training program. 5

20 Chapter 2 Literature Review Individuals depend on aerobic and anaerobic energy systems daily in order to perform increasing amounts of physical exertion (such as activities of daily living, recreational activities, occupational demands, sport, etc). The increasing amount of physical exertion to perform such activities requires an increase in pulmonary oxygen uptake (VO 2 ) to offset the energy requirement of the exercising muscles [24]. Peak voluntary oxygen uptake (VO 2pk ) has previously been shown to be an important cardiopulmonary measurement that is often used to classify fitness levels [1], prescribe exercise intensities [1] and has been used to predict mortality risk [25]. VO 2pk has also been previously shown to be "trainable" as significant increases (~10% - 19%) have been reported subsequent to a variety of cardiovascular training programs [10, 14, 26, 56]. In addition, the increased VO 2pk reported subsequent to cardiovascular training programs has been associated with increases in peak work rate capacity (WR pk ) and muscular endurance (time to exhaustion) [8, 27, 56]. Although VO 2pk is commonly used as a marker of cardiovascular training status, other pulmonary gas exchange markers (such as estimated lactate threshold or the time course for adjustment of VO 2 following a work rate transition) can provide pertinent 6

21 information regarding an individual's cardiovascular training status. For instance, changes in the lactate threshold can be estimated non-invasively during graded exercise tests prior and subsequent to a cardiovascular training program via observing the pulmonary gas exchange data [57, 58]. Typically, a method known as the v-slope method is used to identify the estimated lactate threshold, which is defined as the point in which carbon dioxide production measured at the mouth (VCO 2 ) begins to increase disproportionately to the rise in VO 2 [57, 58]. The ventilatory equivalents (V E /VCO 2, V E /VO 2 ) are ratios of ventilation (V E ) to VCO 2 and to VO 2 and can also be used to help identify the estimated lactate threshold [24]. During exercise within the heavy intensity domain (above lactate threshold, >LT) there is an increased rate of appearance of lactatic acid (HLa) which becomes dissociated into hydrogen ions (H + ) and lactate (La-) [29]. Carbon dioxide (CO 2 ) is one product of the reaction when the H + are buffered by bicarbonate (HCO - 3 ) [24, 29]. The additional CO 2 production from the H + buffering reaction along with the CO 2 production from the tricarboxylic acid (TCA) cycle can be observed in the VCO 2 data during a graded exercise test as a sharp, upward deflection point [24]. As VCO 2 increases there will be an increased ventilatory drive (increasing V E ) and therefore the rise in V E /VO 2 will continue to increase while V E /VCO 2 remains constant indicating isocapnic buffering and the estimated lactate threshold [24]. In addition to VO 2pk and the estimated lactate threshold, further information regarding the cardiovascular training status of an individual can be obtained from observing the time course of the VO 2 response during non-steady state exercise. During a transition from unloaded, or minimally loaded (20 watts), exercise to a work rate within 7

22 the moderate intensity domain (<LT) the VO 2 will follow an exponential pattern, subsequent to a time delay, prior to reaching a newly established steady state [59]. This exponential pattern can be modeled mathematically and will display two phases during the exercise transition within the moderate intensity domain [60]. The phase II of the non-steady state VO 2 response (fundamental phase) has been shown to predominantly reflect the kinetic response of the muscle's oxygen utilization [60, 61]. The time constant of the phase II VO 2 response (τvo 2 ) has been previously reported to become faster during moderate intensity work rate transitions following both traditional cardiovascular training programs [8, 62, 63] and high-intensity interval training (HIIT) programs [8, 56, 62]. Although rather time consuming during the data collection and data analysis processes, determining changes in τvo 2 subsequent to cardiovascular training programs provides more confidence in the results and interpretation of any changes observed in VO 2pk. Since τvo 2 can be assessed within the moderate intensity domain (<LT), the participant does not have to provide as great of effort during the test unlike the maximal effort required during a graded exercise test when assessing VO 2pk. These cardiovascular adaptations that can be observed within the pulmonary gas exchange data are important indicators of the effectiveness of a cardiovascular exercise program and an individual's ability to perform work. The guidelines set forth by the American College of Sports Medicine (ACSM) recommends that a healthy adult participate in 5-7 days of moderate intense (40%-60% VO 2pk ) cardiovascular exercise or 3 days of vigorous intense ( 60%VO 2pk ) cardiovascular exercise each week [1]. The health benefits of regular cardiovascular exercise, such as the previously mentioned ACSM recommendations, include decreased risk for many chronic cardiovascular, 8

23 pulmonary, and metabolic diseases [1-4]. Although research has demonstrated the importance of aerobic exercise in disease prevention [1-4], it was recently reported by the American Heart Association (AHA) that approximately 30% of the adult population within the United States did not engage in regular cardiovascular exercise [2]. The most commonly reported explanation provided by adults as to why they are not able to engage in regular cardiovascular exercise was due to a "lack of time" within their daily routine [5, 6]. Also shockingly noted in the same report issued by the AHA, 18% of girls and 10% of boys (grades 9-12) reported leading sedentary lifestyles by not engaging in physical activity at least one day per week [2]. This is a mortifying statistic as the increased occurrence of sedentary lifestyles often observed within the adult population is also being portrayed within the youth population of the United States. Although the trend for sedentary lifestyles seem to be high in both the youth and adult populations, sedentary lifestyles have been defined as a modifiable risk factor for cardiovascular disease [1]. Therefore it could be suggested that further scientific investigations on the best approach to integrate exercise into a time constrained daily routine and the effectiveness of the exercise programs are of vital importance. Therefore, the primary objectives of this dissertation is to 1) determine the systemic effects of an alternative exercise (blood flow restriction, BFR) technique implemented into a cardiovascular exercise model and 2) to compare the physiological training adaptations from a BFR low-intensity cardiovascular training program to the training outcomes from a high-intensity cardiovascular training program. High-Intensity Interval Training (HIIT) Programs 9

24 High-intensity interval training (HIIT) programs have gained much attention within the exercise science field due to the advantageous cardiovascular training adaptations that have been demonstrated to occur within a relatively short amount of time (2-6 weeks) [7, 9, 64]. HIIT sessions include repeated intermittent high-intensity exercise bouts followed by low intensity exercise bouts [28]. The portions of the HIIT exercise sessions that are performed at high intensities equate to an "all-out effort" [9] and/or near-maximal ( 90% VO 2pk ) [7, 65] efforts. The performance of the high intensity exercise allows some HIIT exercise programs to reduce exercise training volume by up to 80-90% compared to traditional continuous endurance training programs [8, 9]. The drastic reduction in exercise volume, allowing for a relatively low time commitment, and rapid physiological adaptations makes HIIT programs an attractive alternative exercise option for many. Short-Term High-Intensity Interval Training (HIIT) Responses Previous literature has suggested that HIIT improves VO 2pk in both healthy [9, 10, 66] and clinical (i.e., cardiovascular disease, CVD) populations [67-69]. Previously, a HIIT (95% VO 2pk ) program was shown to significantly increase VO 2pk to a greater extent compared to traditional, continuous, moderate intensity (50% VO 2pk ) cardiovascular training, even though the moderate intensity program did significantly increase VO 2pk compared to pre-training levels [70]. However, similar changes in VO 2pk have been reported following HIIT and continuous (65% VO 2pk ) cardiovascular training programs [8] as well as no change in VO 2pk subsequent to a HIIT program [64, 71]. Furthermore, increases in citrate synthase (CS) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) have been shown following both traditional, continuous 10

25 cardiovascular training (5 days/week, 6 weeks, ~2250 kj/week) as well as HIIT (3 days/week, 6 weeks, ~250 kj/week) in a young, healthy population [9]. CS is the enzyme responsible for catalyzing acetyl-coa and oxaloacetate to citrate in the first step of the TCA cycle [29] while PGC-1α has been associated with the promotion of mitochondrial biogenesis [31, 32]. Despite the increased values from the pre-training levels, similar training adaptations occurred subsequent to both cardiovascular training programs (HIIT, continuous) as there was no difference in CS or PGC-1α between the groups during the post-training analysis [9]. In addition, a short-term (2 week), low-volume HIIT program demonstrated increases in CS, phosphocreatine, and creatine kinase activity subsequent to training [10]. The increased CS reported following HIIT [9, 10] may provide a faster transition for oxidative phosphorylation in order to match the energy requirement imposed by the exercising muscles. This could also be supported by the previously observed speeding of the τvo 2 during phase II of a moderate intensity transition following a HIIT program [8, 56]. The culmination of the previous results could suggest that HIIT can help to improve the individual's aerobic capacity, capacity for oxidative phosphoralation [29] and initiate mitochondrial biogenesis [31]. The effect that a HIIT program had upon the oxygenation status of the microvasculature during an incremental fatigue protocol had been previously observed [72]. The authors reported that after six weeks of an HIIT program significant increases in microvascular deoxygenation ([HHb]) during an incremental fatigue protocol was observed [72]. According to the interpretation of the authors, the increased [HHb] subsequent to the HIIT program suggested an improvement in oxygen extraction from the muscle [72]. According to Fick's equation (VO 2 = CO x a-vo 2diff ), if the participants 11

26 ability to extraction oxygen (i.e., a-vo 2diff ) were to increase following training, peak oxygen consumption (VO 2pk ) may be elevated as well. However, no significant difference in [HHb] kinetics were detected during moderate intensity work rate transitions following a HIIT program [8, 56] or a continuous, cardiovascular training program [8]. The [HHb] kinetics have been suggested to reflect the rate of oxygen extraction within the microvasculature [8, 56]. Interestingly, the authors observed a faster response in τvo 2 during phase II of a moderate intensity transition after just two exercise sessions for each of the exercising conditioning (HIIT, continuous) [8]. The authors' conclusion was that during each mode of aerobic exercise that was examined (HIIT, continuous) an increase in muscle oxygen utilization was observed with no changes observed in the rate of oxygen extraction, thus resulting in a matching of oxygen utilization and blood flow following training [8]. These findings [8, 72] help to provide mechanistic evidence to the effect and time course of the training adaptations observed subsequent to HIIT exercise programs [9, 10, 66]. However, further investigation is required to explain how other alternative modes of cardiovascular exercise (i.e., BFR) may influence these training adaptations. Acute Physiological Responses of High-Intensity Interval Training (HIIT) Sessions Participants performing a single HIIT exercise session (6 intervals, 30 seconds allout intensity, 2 minutes active recovery) has been shown to achieve 90% VO 2pk and high levels of blood lactate concentration (15.3 ± 0.7 mmol/l) [65]. The authors attribute these physiological responses to be possible precursors for the training adaptations associated with HIIT exercise [28, 65]. The increase in blood lactate concentration (i.e., high metabolic stress) observed during HIIT exercise [65, 73] have been suggested to be 12

27 a possible precursor for an elevated expression of vascular endothelial growth factor (VEGF) mrna [54]. Vascular endothelial growth factor (VEGF) has been established as a potent exercise-induced angiogenic stimulator [48, 49] and has been associated with the formation of new capillaries and improvements in oxygen delivery to exercising skeletal muscle [49-52]. However, the acute effects that HIIT has upon the microvascular oxygenation and neuromuscular activation of the exercising muscle remain to be investigated. Although HIIT appears as an attractive exercise program due to the reduction in the total exercise volume (i.e., time commitment) and rapid cardiovascular adaptations, the use of high exercise intensities becomes a concern due to the high amounts of effort required by the participants to complete the training program. It would be of interest to investigate the implementation of an alternative exercise technique (i.e., BFR) that could reduce the exercising intensity and determine if significant cardiovascular training adaptations will still occur subsequent to cardiovascular training. Blood Flow Restriction (BFR) Exercise Heavy resistance exercise, utilizing an intensity of 65% of an individual's one repetition maximum (1RM) and/or maximal voluntary contraction (MVC), has traditionally been used in progressive strength training programs to increase skeletal muscle strength and hypertrophy [74, 75]. However, recently an alternative exercise technique known as blood flow restriction (BFR) exercise has gained much attention as low exercise intensities (20% - 40% 1RM/MVC) are utilized to mimic high-intensity exercise responses. Increases in skeletal muscle cross-sectional area [13, 41], muscle strength [13, 17, 40, 41], maximal rate of torque development [40], growth hormone 13

28 concentration [16] and muscle protein synthesis [38, 39] have been demonstrated as training adaptations subsequent to BFR low-intensity resistance exercise. Blood Flow Restriction (BFR) Cardiovascular Training Responses In addition to the muscle hypertrophy and strength improvements associated with BFR resistance training, BFR techniques have been previously incorporated into lowintensity cardiovascular exercise (i.e., walking, cycling) models and have demonstrated advantageous cardiovascular training adaptations subsequent to training. The cardiovascular adaptations include increased VO 2pk [14, 15, 21], muscular endurance (as assessed by an increased time to exhaustion) [14, 20, 21], CS [20, 23], anaerobic capacity as assessed by the Wingate test [15], stroke volume [15], skeletal muscle hypertrophy [13, 14], bilateral dynamic strength (leg press 1RM) [13], unilateral dynamic strength (leg curl 1RM) [13] and isometric strength [13] following training. Although these beneficial cardiovascular adaptations have been previously reported subsequent to BFR cardiovascular exercise programs, many questions remain to be answered regarding the stimulus, or stimuli, responsible for the training outcomes. Acute Physiological Responses of Blood Flow Restriction (BFR) Cardiovascular Exercise A potential mechanism for the increased aerobic capacity subsequent to BFR cardiovascular exercise training programs could be due to a greater mitochondrial density within the trained musculature. Muscle biopsy samples from the vastus lateralis after one session of low-intensity (26 ± 4% of peak work load), unilateral knee extension exercise demonstrated that PGC-1α was significantly greater subsequent to BFR knee extension exercise compared to free-flow (control) exercise [22]. Once again, PGC-1α has been 14

29 suggested to be a vital component during exercise induced mitochondrial biogenesis process [31]. However another potential acute response previously observed during acute BFR exercise is an increased VEGF concentration subsequent to exercise since VEGF has been previously reported as a potent exercise-induced angiogenic stimulator [48, 49]. It has previously been demonstrated that increases in plasma vascular endothelial growth factor receptor 2 (VEGF-R2) concentration occurs following acute low-intensity (20% 1RM) BFR resistance exercise (i.e., knee extension) [53]. While VEGF-R2 mrna has been shown to be significantly elevated within skeletal muscle (i.e., vastus lateralis) following low-intensity (40% 1RM) BFR knee extension exercise [43] and 45 minutes of dynamic, low-intensity knee extension exercise (24 ± 3% peak workload) combine with BFR (50 mmhg lower body positive pressure) [54]. In addition, the authors of the investigation demonstrated a significant, positive correlation (r = 0.54, p < 0.05) between exercise induced VEGF mrna expression and venous lactate concentration, thus suggested that the VEGF mrna expression could be related to the "metabolic stress" accrued from exercise [54]. The observed relationship between VEGF expression and metabolic stress (i.e., lactate concentration) during BFR exercise could potentially explain the elevations in VEGF observed previously during BFR exercise [43, 53] as lactate concentration was also shown to be significantly greater during low-intensity BFR exercise compared to low-intensity control exercise [22, 53, 76] and during exercise under hypoxic breathing conditions [77]. Another potential mechanistic link for the elevations of VEGF following BFR exercise could be due to the increased neuromuscular activation that is associated with 15

30 BFR exercise compared to free-flow (control) exercise [16-19]. VEGF expression has been demonstrated to increase significantly subsequent to increased muscle activity (i.e., motor nerve stimulation) within an animal model [78, 79]. Therefore, it could be suggested that the increased neuromuscular activity observed during BFR exercise [16-19] might be associated with the elevated post-exercise expressions of VEGF that has been previously demonstrated [43, 53]. Despite the uncertainty surrounding the exact mechanistic link for the expression of VEGF during BFR exercise, the concurrent elevation of VEGF expression and increase in PGC-1α (i.e., mitochondrial biogenesis precursor) observed subsequent to BFR exercise may partly be associated with the increases in VO 2pk previously reported subsequent to BFR cardiovascular training [14, 15, 21]. Although the exact mechanism(s) (i.e., hypoxic intramuscular environment [19], metabolic byproduct accumulation [80]) explaining BFR exercise and the resulting training adaptations are currently still under debate, some evidence suggests that the training adaptations (i.e., increased VO 2pk ) following BFR cardiovascular training may occur due to peripheral adaptations [20-23]. This has been previously hypothesized as training adaptations have been observed in the experimental (BFR) leg subsequent to BFR cardiovascular training while no adaptations have been observed in the contralateral, control leg [20-23]. BFR exercise may also follow similar training principles similar to control exercise (i.e., principle of specificity) as performance responses (VO 2pk and time to exhaustion) reported for the experimental condition (BFR) were greater during ischemic testing conditions compared to control testing conditions subsequent to BFR training [21]. These observations come from a series of studies that were conducted 16

31 using lower body positive pressure (LBPP) to provide an ischemic intramuscular environment during exercise [20-23]. The LBPP technique has been shown to reduce blood flow by approximately ~16% (0.3 liters per minute, L/min) during low-intensity exercise [81], while BFR (i.e., proximal occlusion cuff, 130% SBP occlusion pressure) techniques have been shown to reduce blood flow by approximately ~30% (~0.2 L/min) during low-intensity exercise [53]. LBPP (50 mmhg above atmospheric pressure) lowintensity exercise has also demonstrated to significantly increase oxygen extraction (i.e., greater arterial-venous oxygen difference) [81] and exercising heart rate [21] compared to control (i.e., free flow) during acute (one session) low-intensity exercise. Subsequent to LBPP low-intensity cycling training significant increases in VO 2pk [21], muscular endurance (time to exhaustion) [21, 23] and CS [23] were reported when compared to control low-intensity cycling training, similar training responses as BFR cardiovascular training programs. In conclusion, similar training adaptations have been reported following both HIIT and BFR cardiovascular training programs which included significantly increased CS, PGC-1α, and VEGF expression subsequent to acute exercise sessions and improvement in VO 2pk following training. However, to the author's knowledge it has yet been determined how BFR cardiovascular training programs affect the estimated lactate threshold and the oxidative phosphorylation capacity of the trained musculature (i.e., phase II τvo 2 during a moderate intensity step work rate transition). Furthermore, it has been previously suggested that BFR cardiovascular training adaptations result from peripheral adaptations [20-23], therefore, further investigation into the acute effects of BFR on microvascular oxygenation and neuromuscular activation of the skeletal muscle 17

32 could provide further evidence to suggest a potential stimulus/stimuli responsible for the training adaptations observed following BFR cardiovascular exercise programs. Blood Flow Restriction (BFR) Exercise Safety Considerations The beneficial training adaptations that have been demonstrated following the use of BFR training programs have the potential to be applied to a variety of populations (i.e., healthy, novice, elderly, rehabilitation, occupational, sport), however, safety of BFR exercise is of primary importance as formation of thrombus and induced microvascular damage has been associated with increased pressurization of blood vessels [82-85]. Therefore, previous studies have investigated potential safety concerns (i.e., blood clot formation, microvascular inflammation, microvascular damage, neuromuscular complications, muscle damage) that have been raised while using BFR exercise techniques. For instance, Clark et al [86] previously demonstrated that acute (four weeks, 3 days/week) knee extension training within a young, healthy population (18-30 years old) resulted in an increase in isometric strength (BFR = ~8%, high-intensity = ~13%) with no difference observed in ankle-brachial index or nerve conduction (i.e., defined as the latency response of the H-relfex) following the completion of training in either group (BFR or high-intensity). Furthermore, no change acutely (following the first exercise session) or chronically (following the completion of the four week training program) was observed in prothrombin time, fibrinogen, D-dimer, and/or high sensitivity C-reactive protein from baseline (pre-training) measurements in either group [86]. In agreement with Clark et al [86], no change in D-dimer was observed following a BFR knee extension exercise session in young (25.1 ± 2.8 years), healthy males [87] or in stable, older (57 ± 6 years), ischemic heart disease patients [88]. Madarame et al [88], 18

33 also observed no change in high sensitivity C-reactive protein following BFR knee extension exercise in ischemic heart disease patients, a similar finding demonstrated in young, healthy population by Clark et al [86]. The collective conclusion of the previous investigations suggest that BFR exercise does not expose the participant to any higher risk of blood clots or inflammation compared to high-intensity resistance training [86]. Karabulut et al [89] found no significant increase in creatine kinase and/or interleukin 6 following six weeks of resistance training (BFR group and high-intensity group) performed by healthy, older (56.6 ± 0.6 years) males. The authors of the previous investigation suggest that this finding implies a lack of skeletal muscle damage and inflammatory response following BFR exercise training. However, this conclusion should be interpreted with caution since the post-training blood samples were collected 24 hours subsequent to the last training session and creatine kinase has been shown not to peak until hours post exercise [89-91]. Nevertheless, torque decrements (used as an indirect muscle damage marker) following BFR exercise have been shown to return to baseline measurements 24 hours after the exercise session, suggesting a lack of muscle damage in this condition [92]. Furthermore, based upon survey results of 12,642 participants, BFR exercise has been shown to be a safe and effective form of exercise that has been associated with only a small number of complications reported [82]. The most common complication that has been reported during BFR exercise has been subcutaneous hemorrhage or bruising (~13%) of the exercising limb [82]. The results of the survey also demonstrate that BFR exercise sessions typically last 5-30 minutes in duration and are performed on average 1-3 days/week [82]. 19

34 Chapter 3 Effects of Blood Flow Restriction Duration on Muscle Activation and Microvascular Oxygenation During Low-Volume Isometric Exercise Cayot TE, Lauver JL, Silette CR, Scheuermann BW. (2015). Effects of blood flow restriction duration on muscle activation and microvascular oxygenation during lowvolume isometric exercise. Clin Physiol Funct Imaging (Ahead of Print) Introduction Heavy resistance exercise, utilizing an intensity of 65% of an individual's maximal voluntary contraction (MVC), has typically been used in progressive strength training programs to increase skeletal muscle strength and hypertrophy [74, 75]. However, high intensity exercise may not be well suited for use in some patient populations and/or settings (i.e., novice exerciser, elderly, rehabilitation) thereby limiting the potential health and performance improvements [93]. Recent studies have suggested that utilizing blood flow restriction (BFR) techniques in combination with low-intensity (20%-40%MVC) resistance exercise results in similar increases in skeletal muscle crosssectional area [13, 41], muscle strength [13, 17, 40, 41], maximal rate of torque development [40], growth hormone concentration [16] and muscle protein synthesis [38, 39] compared to heavy resistance exercise ( 65% MVC). The proposed mechanisms by which BFR resistance training is thought to work include a hypoxic intramuscular 20

35 environment [19], increased cellular swelling [80], and/or increased metabolic byproduct accumulation [80]. Investigators have previously demonstrated that four weeks of low-intensity (40%MVC) isometric BFR training significantly improved isometric muscle strength and maximal rate of torque development [40]. However, the potential effect that isometric BFR exercise had on neuromuscular activation, both immediately and following training, was not previously examined in that investigation [40]. Although the underlying mechanism(s) explaining BFR training adaptations are still a matter of debate, many studies have shown an increase in neuromuscular activation [16-19] and firing frequency [19] of the exercising muscles during isotonic exercise with BFR conditions compared to free flow (i.e., non-bfr, control) conditions. Increased neuromuscular activation has been hypothesized as a mechanism for eliciting muscular strength gains [40, 94]. Therefore, BFR exercise, which has been shown to increase neuromuscular activation acutely during exercise with low-intensity resistance loads, could potentially provide an alternative approach to heavy resistance training in order to promote muscular strength gains in populations where heavy resistance exercise may not be appropriate. In order for BFR exercise to be an effective exercise modality, the methodological approach to BFR exercise techniques must be systematically evaluated. This includes identifying the most effective occlusion duration, as occlusion durations have varied in previous BFR studies and has been shown to affect cellular swelling [80] and neuromuscular activation [16-19]. For instance, a previous investigation has indicated that an initial restrictive pressure applied four minutes prior to the application of the target occlusion pressure caused a significant decrease in the microvascular oxygenation 21

36 of the skeletal muscle during supine rest as measured via near-infrared spectroscopy (NIRS) techniques [95]. However, this investigation [95] did not examine the potential effect(s) that an initial restrictive pressure could have on microvascular oxygenation and/or neuromuscular activation during exercise under BFR conditions. As previously suggested, if decreased microvascular oxygenation provides a stimulus for altered neuromuscular activation within the muscle [16, 96, 97], then determining the optimal occlusion procedure would be beneficial for developing an effective and efficient BFR exercise program. In order to better understand how neuromuscular activation and microvascular oxygenation is immediately affected by varied BFR occlusion durations, a low-volume isometric exercise protocol was employed during the present investigation. The lowvolume isometric BFR exercise protocol used in the present investigation has not been associated with an increase in muscle strength or hypertrophy and differs from the typical high-volume, isotonic BFR exercise protocols previously reported [98, 99]. However, it was the intent of the present study to primarily focus on the immediate effects that varied occlusion durations had upon neuromuscular activation and microvascular oxygenationdeoxygenation while simultaneously minimizing the confounding influence of the accumulation of metabolic byproducts that would be elicited from a higher exercise volume. Therefore, the primary purpose of the present investigation was to examine the effects of occlusion duration on neuromuscular activation and microvascular oxygenation during BFR isometric knee extension exercise preformed at various sub-maximal intensities. It was hypothesized that a longer occlusion duration would elicit an increased neuromuscular activation at a given sub-maximal contraction intensity. It was also 22

37 hypothesized that a longer occlusion duration would cause a lower microvascular oxygenation response (i.e., greater deoxygenation, deoxy-[hb+mb]) while simultaneously increasing blood volume (i.e., total hemoglobin concentration, [THC]) within the microvasculature as measured using near-infrared spectroscopy Methods Healthy, recreationally active, college-aged male subjects (n = 7; age = 24.8 ± 1.4 years; height = ± 3.0 cm; weight = 81.4 ± 0.1 kg) participated in the study. The number of subjects included in the present investigation was determined based upon previous BFR investigations that observed similar outcome variables [19, 95]. The purpose, benefits, and risks associated with the study were explained to each subject. Prior to participation each subject read and voluntarily signed an informed consent form approved by the university's institutional research review board for human subjects, which was in agreement with the guidelines set forth by the Deceleration of Helsinki. Each subject completed medical health history and activity level questionnaires, which were used for inclusion/exclusion purposes. Any individual that had been previously diagnosed with a metabolic, pulmonary and/or cardiovascular disease (including hypertension) and/or an orthopedic-related injury within the last 12 months was excluded from participation in the study. Each subject was asked to refrain from participating in any strenuous physical activity for 24 hours prior to each testing session. The testing session was scheduled for approximately the same time of day for each subject. Exercise Protocol Each subject participated in four separate sessions, during which one set of four repetitions of sub-maximal isometric knee extension were performed at 90 of knee 23

38 flexion for each BFR condition. The exercise intensities (20%MVC, 40%MVC, 60%MVC, 80% MVC) and BFR conditions were randomized between and within each of the four sessions, respectively. The BFR conditions that were evaluated included control (CON, no occlusion), immediate occlusion (IO) and pre-occlusion (PO). During the CON condition the occlusion cuff was placed around the distal thigh but was not inflated either before or during the exercise. The occlusion cuff was inflated immediately prior to exercise and remained inflated during exercise for the IO condition (~110 seconds). During the PO condition, the occlusion cuff was inflated five minutes prior to the onset of exercise and remained inflated throughout exercise (~7 minutes). The occlusion cuff was immediately deflated following the completion of the last repetition during both of the BFR (IO, PO) exercise sets. At the beginning of each session the subject was seated and remained at rest for five minutes prior to measuring blood pressure. All blood pressure measurements were obtained with the upper arm supported at heart level with the subject seated in an upright position. Following the blood pressure measurement, the distance from the anterior superior iliac spine (ASIS) to the superior border of the patella was measured and recorded. An occlusion cuff (Hokanson, SC5, Bellevue, WA, 6.0 cm width) was positioned at 33% of the distance distal to the ASIS on the dominant thigh, which is similar to the cuff positioning described in a previous BFR investigation [100]. The external occlusion pressure used during all BFR conditions was equivalent to 130% of the subject's resting systolic blood pressure (130% SBP), consistent with the external occlusion pressure used in previous BFR investigations [15, 36, 42, 53, 101]. Surface electromyography (semg) and near-infrared spectroscopy (NIRS) techniques were used 24

39 to measure neuromuscular activation and microvascular oxygenation of the exercising muscle tissue, respectively. Following the placement of the occlusion cuff and all data collection electrodes (see Surface Electromyography Techniques and Near-Infrared Spectroscopy Techniques below), the subject remained seated upright in a padded chair while a padded strap was secured around the ankle of their dominant leg. All exercise was performed on the subject s dominant leg [102]. The subject was secured with straps across the hips and shoulders in order to minimize movement during the isometric exercise sets. The ankle strap was connected to a force transducer (Omega, LCCA-1K, Stamford, CT) so that force production could be recorded and visually displayed to the subject on a computer monitor. The length of the ankle strap was adjusted so that the subject's knee remained at 90 of knee flexion during each isometric contraction. The force transducer was calibrated via the manufacture's guidelines with a five point calibration curve prior to each testing session. The subject performed two sets of eight repetitions of isometric knee extension at self-selected intensities under CON conditions in order to become familiarized with the exercise protocol. Self-selected intensities were used during the familiarization sets so that the subject could practice the intended duty cycle consisting of a five second isometric contraction followed by thirty seconds of recovery. The subject was provided with five minutes of recovery following each of the familiarization sets. The self-selected intensities were recorded during the first visit and used for subsequent warm-up sets throughout the remainder of the investigation. Following the completion of the familiarization/warm-up sets, each subject completed three maximal voluntary isometric contractions (MVIC) at 90 of knee 25

40 flexion. Each MVIC was performed under CON conditions consisting of a five second contraction duration followed by five minutes of recovery. Subsequent to the MVIC, the subject completed one set of four repetitions of sub-maximal isometric knee extension for each BFR condition. In order for the subject to reach the desired sub-maximal intensity a line was placed on the computer monitor to indicate to the subject, via visual feedback, the force needed for each target sub-maximal intensity. The sub-maximal target intensities (20%MVC, 40%MVC, 60%MVC, 80%MVC) were randomized between test days, while the BFR conditions (CON, IO, PO) were randomized between each exercise set within each test day. Each contraction was held for five seconds at the target intensity with 30 seconds of recovery between each repetition. Subjects were provided with 15 minutes of recovery following each set of exercise. Surface Electromyography (semg) Techniques Surface electromyography (semg) was used to examine the neuromuscular activation of the vastus lateralis (VL-RMS) and vastus medialis (VM-RMS) during each contraction. Prior to placement of the semg electrodes, the skin was shaved and cleansed with an alcohol pad. Double differential semg electrodes (Delsys, Bagnoli 8- Channel System, Boston, MA) with a fixed inter-electrode distance of 1 cm were placed on the vastus lateralis and vastus medialis of the dominant thigh. The vastus lateralis semg electrode was placed at an oblique angle approximately 3-5 cm proximal to the patella just lateral to the midline over the muscle belly [103]. The vastus medialis semg electrode was placed at an oblique angle approximately 2 cm medially from the superior border of the patella within the distal third of the vastus medialis muscle belly [103]. The 26

41 raw semg signal was collected at a sampling frequency of 2,000 Hz using LabChart 7.0 (ADInstruments, Inc., Colorado Springs, CO). Near-Infrared Spectroscopy (NIRS) Techniques Frequency-domain multi-distance (FDMD) near-infrared spectroscopy (NIRS) was used to continuously monitor absolute deoxy-hemoglobin (deoxy-[hb+mb]) and oxy-hemoglobin (oxy-[hb+mb]) responses. Total hemoglobin concentration ([THC]), an indicator of blood volume within the microvasculature, was calculated as the sum of the deoxy-[hb+mb] and oxy-[hb+mb] signals. The NIRS system (Oxiplex TS, ISS, Champaign, IL) was calibrated prior to each data collection session according to the specifications provided by the manufacturer and was operated at wavelengths of 690 nm and 830 nm with a sampling frequency of 2 Hz. It has been previously reported that the deoxy-[hb+mb] signal is less sensitive to blood volume changes (Δ[THC]) compared to the oxy-[hb+mb] signal [104] and that deoxy-[hb+mb] can be used as an estimation of oxygen extraction [105, 106]. Although the microvascular deoxygenation response within the vastus lateralis has been shown to be non-uniform throughout the muscle during fatiguing exercise, the greatest microvascular deoxygenation response has been observed at the middle region of the vastus lateralis during knee extension exercise compared to the distal regions [107]. Therefore, the NIRS sensor was positioned midway between the ASIS and the superior border of the patella over the muscle belly of the vastus lateralis in the present investigation. Prior to placement of the NIRS sensor the skin was shaved and cleansed with an alcohol pad. The NIRS sensor was covered with a dark black cloth, in order to prevent stray visible light sources from affecting the data acquisition, and secured with straps placed over the vastus lateralis muscle. 27

42 Data Processing The highest MVIC during the first testing session was used to calculate all submaximal intensities (20%MVC, 40%MVC, 60%MVC, 80%MVC) throughout the entire investigation. All semg and NIRS data were analyzed during the middle three seconds of each five second isometric contraction. The raw semg data were amplified by a gain of 1,000 (Bagnoli 8-Channel System, Delsys, Boston, MA), digitally filtered using a band-pass filter (10Hz-500Hz) and then smoothed using a 50 millisecond root means squared (RMS) window (LabChart 7.0, ADInstruments, Inc., Colorado Springs, CO). The semg data was then normalized to an average of the semg data collected during all three CON MVIC during each testing session and therefore, all semg data are represented as a percentage of the MVIC (%MVIC). All exercising NIRS data were normalized using a "physiological calibration", as previously described [105]. After the completion of all exercise, external occlusion (250 mmhg) was applied to the distal thigh for approximately five minutes until the deoxy- [Hb+Mb] and oxy-[hb+mb] signals reached plateaus. All NIRS data are expressed as a percentage of the maximal deoxygenated plateau (%DP) according to the physiological calibration [105]. Statistical Analysis To determine if there were any significant differences in neuromuscular activation of the vastus lateralis (VL-RMS) and/or vastus medialis (VM-RMS) during exercise a two-way (BFR condition x sub-maximal intensity) analysis of variance (ANOVA) with repeated measures was used. Two-way (BFR condition x sub-maximal intensity) ANOVA with repeated measures was also completed for the microvascular 28

43 deoxygenation response (deoxy-[hb+mb]) and total hemoglobin concentration response ([THC]) during exercise. The target sub-maximal isometric force and produced submaximal isometric force during the isometric contractions were examined via a two-way (force x sub-maximal intensity) ANOVA with repeated measures in order to determine significant differences. All significant ANOVA results were followed by Tukey's post hoc analysis in order to identify the specific differences. For all statistical comparisons, significance was set a priori at p All data are presented as mean ± SD. Statistical analysis was completed using Sigma Stat software (Sigma Stat 3.5, Systat Software, San Jose, CA) Results Resting Blood Pressure & Applied Occlusion Pressure The mean resting systolic blood pressure was 124 ± 10 mmhg, while the mean resting diastolic blood pressure was 81 ± 4 mmhg. Therefore, the mean applied occlusion pressure (130% SBP) used during this investigation was 162 ± 12 mmhg. Isometric Force Results No significant difference was found between target sub-maximal isometric force and the generated sub-maximal isometric force at any of the sub-maximal contraction intensities. Surface Electromyography (EMG) Results Although there was a significant increase in neuromuscular activation of both the VL-RMS and VM-RMS with increasing sub-maximal exercise intensities (p < 0.001), there was no difference in VL-RMS or VM-RMS between any of the BFR conditions during any sub-maximal intensity (Figure 1 & Figure 2). 29

44 Near-Infrared Spectroscopy (NIRS) Results When examined relative to the maximal deoxygenated plateau (DP), significant differences were detected for deoxy-[hb+mb] (Figure 3) and [THC] (Figure 4) based upon BFR condition. PO (105.4 ± 36.2%DP) resulted in a higher deoxy-[hb+mb] compared to both CON (81.5 ± 28.8%DP) and IO (94.8 ± 30.7%DP) at a sub-maximal intensity of 20%MVC (p < 0.001, p = 0.043). Furthermore, deoxy-[hb+mb] was also significantly higher during IO compared to CON at 20%MVC (p = 0.009). At a submaximal intensity of 40%MVC, deoxy-[hb+mb] was greater during both PO (107.5 ± 20.1%DP, p <0.001) and IO (100.1 ± 14.0%DP, p = 0.014) compared to CON (87.4 ± 17.9%DP). During sub-maximal intensities of 60%MVC (p = 0.01) and 80% MVC (p = 0.007), deoxy-[hb+mb] was higher during PO (116.7 ± 19.8%DP, ± 14.5%DP) compared to CON (103.7 ± 23.6%DP, ± 16.7%DP). PO (101.6 ± 19.7%DP, p < 0.001) and IO (98.6 ± 21.2%DP, p < 0.001) elicited higher [THC] compared to CON (91.0 ± 20.1%DP) at a sub-maximal intensity of 20%MVC. During exercise at 40%MVC, [THC] was greater during both PO (97.8 ± 9.6%DP, p < 0.001) and IO (96.6 ± 9.6%DP, p < 0.001) compared to CON (88.2 ± 7.9%DP). [THC] was higher during PO (102.5 ± 13.9%DP, p < 0.001) and IO (98.9 ± 12.2%DP, p = 0.044) compared to CON (95.0 ± 13.0%DP) at a sub-maximal intensity of 60%MVC. PO (90.9 ± 6.9%DP, p = 0.004) and IO (90.2 ± 8.6%DP, p = 0.012) resulted in higher [THC] at a sub-maximal intensity of 80%MVC compared to CON (85.4 ± 6.0). There were no differences in [THC] between PO and IO BFR conditions at 20%MVC, 40%MVC, 60%MVC, or 80%MVC. 30

45 3.4 - Discussion In contrast to our initial hypothesis, the results of the present investigation demonstrated no significant changes in neuromuscular activation during low-volume, isometric exercise between BFR and CON conditions. More specifically, differing BFR occlusion durations (immediate occlusion, IO; pre-occlusion, PO) did not elicit an immediate substantial impact on neuromuscular activation during low-volume, isometric exercise at a variety of sub-maximal exercise intensities (20%MVC, 40%MVC, 60%MVC, 80%MVC). In partial agreement with our second hypothesis, a longer occlusion duration (PO) resulted in a greater deoxy-[hb+mb] response than immediate occlusion (IO), however this was only observed at a low exercise intensity (20% MVC). Although PO resulted in a greater deoxy-[hb+mb] response compared to CON at all tested exercise intensities, IO only elicited in a greater deoxy-[hb+mb] response compared to CON at low exercise intensities (20% MVC, 40%MVC). This observation could suggest that the exercise-induced metabolic stress, as measured by NIRS, during IO exercise is attenuated at high exercise intensities (60% MVC, 80% MVC). In addition, the present investigation revealed that BFR (IO, PO) exercise caused higher microvascular [THC] compared to CON exercise, however no difference in [THC] was observed between the BFR (IO, PO) conditions at any sub-maximal intensity. The conflicting results regarding neuromuscular activation observed during the present investigation and the previous BFR isometric investigation [19] could be due to differences in exercise volume and/or applied external occlusion pressures. During the previous investigation [19] the duty cycle included a two second isometric contraction at a sub-maximal intensity of 20%MVC followed by a two second recovery, this exercising 31

46 duty cycle (i.e., two second contraction, two second recovery) was continued for a duration of four minutes. During the present investigation the exercise volume was considerably lower as each subject performed three sets of four isometric repetitions. The duty cycle of the present investigation included a five second contraction duration followed by 30 seconds of rest between each isometric repetition. Unlike the highvolume exercise protocols typically used in previous BFR investigations [98, 99], a low exercise volume was chosen for the present investigation in an attempt to negate the influences that fatigue and/or increases in metabolic accumulation may have upon neuromuscular activation. Therefore, the greater exercise volume that was performed in the previous BFR isometric investigation [19] could have caused an increase in metabolic accumulation, thus possibly leading to increased neuromuscular activation [108] that has typically associated with BFR exercise [16-19]. It is also important to note that the external occlusion pressures utilized during the BFR conditions throughout the two studies were different. This could have potentially affected the neuromuscular activation outcome by creating a difference in the hypoxic intramuscular environment, as suggested previously [19]. The previous investigation [19] utilized an absolute occlusion pressure (200 mmhg) for all subjects that was higher than the relative occlusion pressure used in the present investigation which was determined by the subject's resting systolic blood pressure (130% SBP, 162 ± 12 mmhg). Similar neuromuscular activation between BFR and CON conditions, as observed in the present investigation, have been demonstrated during isotonic BFR exercise elsewhere [109]. During the previous investigation [109] subjects performed three sets of isotonic knee extension exercise at a sub-maximal intensity of 30%MVC to volitional 32

47 fatigue. The absolute occlusion pressure of 100 mmhg was applied at the beginning of the first exercise set and released after the completion of the third exercise set. The occlusion cuff remained inflated while the subject received 45 seconds of intermittent recovery following the completion of each exercise set and therefore occlusion duration was dictated by the number of repetitions performed to volitional fatigue. Although not specified by the author, the number of repetitions performed to volitional fatigue under BFR conditions decreased during subsequent exercise sets throughout the session, however, no changes in neuromuscular activation were observed during the concentric phase between the initial BFR exercise set and the fatiguing BFR exercise sets [109]. The combined findings of the previous investigation [109] and the present investigation suggest that occlusion duration may not directly influence neuromuscular activation during BFR exercise. However, high exercise volume and/or the magnitude of applied external occlusion pressure used may elicit the higher neuromuscular activation commonly observed with BFR exercise [16-19]. Previous investigations [97, 108] provides support that increases in neuromuscular activation demonstrated via BFR exercise compared to CON exercise could not only be dependent on reduced skeletal muscle oxygenation, as previously hypothesized [19], but also be affected by intramuscular metabolic stress [80]. Increases in metabolic stress could be due to the diminished venous return commonly associated with BFR exercise [53, 110] thus resulting in the accumulation of metabolites and blood volume within the exercising musculature. Therefore, in partial agreement with our secondary hypothesis and a previous investigation performed with subjects at rest in the supine position [95], the presence of occlusion (IO, PO) elicited greater deoxy-[hb+mb] 33

48 compared to CON while performing isometric exercise at various sub-maximal intensities, suggesting a greater oxygen extraction [105, 106]. However, the deoxy- [Hb+Mb] was not significantly different between IO and CON at 60%MVC and 80%MVC intensities. This result could indicate that an exercise intensity threshold exists, in which, the difference in metabolic demand (i.e., deoxy-[hb+mb]) between IO and CON exercise is attenuated due to an increasing demand during high intensity (60%MVC, 80%MVC) exercise [111]. It has been previously demonstrated that there are no additive benefits (i.e., muscular hypertrophy and strength) for the use of BFR techniques during high-intensity exercise programs [112]. The attenuated deoxy- [Hb+Mb] response observed during the present investigation may support the previous finding [112]. In accordance with our secondary hypothesis, [THC] was greater during occluded (IO, PO) exercise compared to CON exercise in the present investigation. This observation could be explained by an accumulated blood volume within the area of interrogation [113, 114], possibly due to the lack of venous return commonly associated with BFR exercise [53, 110]. Although we can only speculate the change in intramuscular metabolic stress during the present investigation, the findings of the present investigation are consistent with an increased demand for oxygen (increased deoxy- [Hb+Mb]) and/or accumulation of blood volume (increased [THC]) within the exercising musculature. Despite these observations, the metabolic stress during the present investigation (low-volume, isometric exercise) may not have reached the theoretical threshold required to affect neuromuscular activation, as previously suggested [108]. The increased [THC] observed in the present investigation is in agreement with the 34

49 phenomenon that has been previously reported in BFR literature known as "cellular swelling" [80]. Cellular swelling has been associated with increases in protein synthesis [115, 116], which has also been suggested to be a BFR training adaptation [38, 39]. However, the authors of the present investigation caution this immediate explanation, as the increased [THC] could also reflect the accumulation of blood in adjacent vessels/capillaries that were not fully recruited prior to the occlusion and/or muscle contraction. During the present investigation surface electromyography was only recorded from the vastus lateralis and vastus medalis muscles during exercise. This is a limitation to the present investigation as possible compensatory increases in neuromuscular recruitment of other synergistic muscles [117] were not accounted for. Furthermore, microvascular deoxygenation responses were only obtained at one point within the vastus lateralis, while a non-uniform microvascular response to fatiguing exercise has been previously documented [107]. However, it is important to note that the NIRS sensor placement remained consistent throughout each exercise session for each subject in order to reduce the within-subject variability between sessions. However, future investigations should consider the effect of varied occlusion pressures on synergistic muscle groups as well as the microvascular oxygenation response at multiple sites in the vastus lateralis and other muscles Conclusion The primary purpose of this investigation was to observe if neuromuscular activation and microvascular oxygenation was immediately affected from the application of varying BFR occlusion durations. The exercise volume remained low compared to 35

50 previous BFR investigations [98, 99] in an attempt to negate the effects of fatigue and/or metabolic byproduct accumulation on neuromuscular activation. In conclusion, neuromuscular activation does not seem to be affected solely from the application of external occlusion durations (i.e., IO and PO) but possibly due to increased metabolic stress (i.e., high applied external occlusion pressure, high exercise volume and short recovery periods). PO exercise elicited a greater deoxy-[hb+mb] response compared to IO exercise at an exercise intensity of 20% MVC. In addition, the increased deoxy- [Hb+Mb] response during IO exercise compared to CON exercise was only observed during low-intensity exercise (20% MVC, 40% MVC). These findings could suggest that the effect of the varied BFR conditions may be attenuated during higher intensity exercise. Furthermore, all BFR conditions displayed a greater [THC] during exercise compared to CON exercise, thus indicating the presence of an increased microvascular blood volume within the area of interrogation. According to the accumulated results of previous BFR investigations and the present investigation it is suggested that BFR occlusion duration alone may not be the primary stimulus for the increase in neuromuscular activation commonly associated with BFR exercise. Furthermore, providing an adequate metabolic stress (i.e., high applied occlusion pressure, high exercise volume, and/or short recovery periods) may be a driving mechanistic link for the observed health and performance improvements associated with BFR exercise. 36

51 Figure 3-1 No significant difference in neuromuscular activation of the VL-RMS was observed between blood flow restriction conditions at any of the sub-maximal intensities (p > 0.05). 37

52 Figure 3-2 No significant difference in neuromuscular activation of the VM-RMS was observed between blood flow restriction conditions at any of the sub-maximal intensities (p > 0.05). 38

53 Figure 3-3 Significantly higher deoxygenated hemoglobin (deoxy-[hb+mb]) was observed with immediate occlusion (IO) and pre-occlusion (PO) blood flow restriction conditions during low-intensity (20%MVC, 40%MVC) exercise compared to control (CON) exercise. Deoxy-[Hb+Mb] was significantly higher with PO compared to IO during exercise at 20%MVC. Significantly higher deoxy-[hb+mb] was observed during high-intensity (60%MVC, 80%MVC) exercise with PO compared to CON. *Significantly different compared to CON, p #Significantly different compared to IO, p

54 Figure 3-4 A higher total hemoglobin concentration ([THC]) was observed during the immediate occlusion (IO) and pre-occlusion (PO) blood flow restriction conditions compared to the free-flow (control, CON) condition. *Significantly different compared to CON, p

55 Chapter 4 Acute Effects of Blood Flow Restriction During Heavy Intensity Cycling Exercise Introduction Blood flow restriction (BFR) training using low-intensity loads (20-40% one repetition maximum, 1RM) have been previously shown to increase skeletal muscle strength [17] and hypertrophy (i.e., increased cross-sectional area) [17, 37] within resistance training models. Furthermore, increases in vascular endothelial growth factor receptor 2 (VEGF-R2) concentrations have been previously reported following BFR lowintensity knee extension exercise at exercise intensities of 20% 1RM [53] and 40% 1RM [43]. VEGF-R2 has been established as a potent exercise-induced angiogenic stimulator [48] and has been associated with the formation of new capillaries and improvements in oxygen delivery to exercising skeletal muscle [50-52]. Additionally, previous investigations have demonstrated advantageous cardiovascular training adaptations subsequent to BFR low-intensity cardiovascular (i.e., walking, cycling) training, resulting in increased peak oxygen uptake (VO 2pk ) [14, 15, 21], muscular endurance (time to exhaustion) [14, 20, 21], citrate synthase (CS) [20] and anaerobic capacity (i.e., Wingate test) [15]. Significant increases in skeletal muscle strength [13] and hypertrophy [13, 14] 41

56 have been demonstrated concurrently with the cardiovascular adaptations following BFR low-intensity cardiovascular training. BFR low-intensity cardiovascular training may be a favorable alternative exercise approach for populations with a lowered exercise tolerance due to the advantageous cardiovascular adaptations, concurrent skeletal muscle strength and hypertrophy increases and relatively low exercise intensities used during training. However, despite the beneficial outcomes associated with BFR cardiovascular training, the physiological mechanism(s) responsible for the training adaptations are still under debate. Therefore it is imperative to systematically evaluate the physiological effects of BFR low-intensity cardiovascular exercise so that an effective exercise prescription may be created for BFR cardiovascular training programs. For instance, an acute bout of BFR cycling exercise has been shown to decrease exercising stroke volume while increasing exercising heart rate at a variety of submaximal cycling intensities (20% VO 2pk, 40% VO 2pk, 60%VO 2pk ) [45]. During this perturbation of exercising stroke volume and heart rate, cardiac output is maintained similar to a free-flow (control) exercise levels measured at the same sub-maximal cycling intensities [45]. The lowered exercising stroke volume during BFR cycling exercise may be due to the diminished venous return commonly associated with venous pooling within the muscle during BFR exercise [53, 110]. Although, exercising stroke volume and heart rate have been shown to be affected by BFR techniques, exercising oxygen uptake measured at the mouth (VO 2 ) was not significantly different during sub-maximal BFR and free-flow (control) cycling exercise [45]. However, exercising VO 2 has been shown to be significantly higher during BFR walking compared to free-flow (control) walking 42

57 exercise [13]. According to Fick's equation, the higher exercising VO 2 observed during BFR walking [13] could be due to an increased oxygen extraction (A-VO 2diff ) previously demonstrated during BFR exercise [81], despite similar cardiac output values [45]. It has also been previously proposed that the cardiovascular adaptations observed subsequent to a BFR training program may be due to peripheral adaptations [20-23]. However, little information is known how BFR cardiovascular exercise affects the neuromuscular activation and microvascular oxygenation of the exercising muscles. Therefore, the primary purposes of the present investigation was 1) to determine the metabolic, neuromuscular activation, and microvascular oxygenation responses to an acute bout of BFR cycling exercise at a variety of occlusion stimuli (control, low, high) and 2) to evaluate the subsequent effect on VEGF-R2 concentrations following a single BFR cycling. Varied occlusion stimuli (control, low, high) were used in the present investigation to determine the acute metabolic, neuromuscular activation, and microvascular oxygenation responses to perturbations in intramuscular hypoxia and metabolite accumulation. The primary hypothesis was that a suprasystolic (high, HO) occlusion pressure would elicit a greater microvascular deoxygenation (deoxy-[hb+mb]) response and higher neuromuscular activation during BFR exercise compared to control (CON) exercise, as measured using near-infrared spectroscopy (NIRS) and surface electromyography (semg) techniques, respectively. The secondary hypothesis was that a greater plasma VEGF-R2 concentration would occur following HO cycling exercise compared to CON exercise. 43

58 4.2 - Methods Eight healthy, recreationally active, male subjects participated within the present research investigation. The purpose, benefits, and risks associated with participation were explained and each subject read and voluntarily signed an informed consent form that was in agreement with the Deceleration of Helsinki and approved by the university's institutional review board for human subjects prior to participation. Each subject completed medical health history and activity level questionnaires and the answers to the questionnaires were used for subject inclusion and exclusion criteria. Any subject that had previously been diagnosed with a metabolic, pulmonary, and/or cardiovascular disease, including hypertension, was excluded from the investigation. In addition, any subject that had been diagnosed with deep vein thrombosis, any blood clotting disorder, and/or an orthopedic-related injury (within the last 12 months) was also excluded from participation within the investigation. Each subject was asked to refrain from participating in any strenuous physical activity 24 hours prior to each data collection session. Each session was scheduled at approximately the same time of day for each subject with at least 48 hours between each subsequent visit. Exercise Testing During the first visit, anthropometric measurements were recorded including age (years), height (meters, m), weight (kilograms, kg), resting blood pressure (mmhg), and body composition (percent body fat, %fat). At the beginning of each session the subject was seated and remained at rest for five minutes prior to measuring blood pressure. All blood pressure measurements were obtained with the upper arm supported at heart level with the subject seated in an upright position. The total body composition was 44

59 determined by utilizing an air displacement method known as the BodPod Body Composition System (BodPod Express EX, Cosmed, Rome, Italy). All subject demographic data are displayed in Table 1 below. Next each subject completed an incremental ramp test to volitional fatigue on a stationary cycle ergometer (Excalibur Sport, Lode, The Netherlands) using a forcing function of 20 watts per minute (W/min). During the progressive ramp exercise test the subjects were asked to maintain a pedaling cadence of revolutions per minute (rpm). The exercise test was terminated when the pedaling cadence fell below 50 rpm in spite of strong verbal encouragement or the subject achieved volitional fatigue. Pulmonary gas exchange was recorded breath by breath using a commercially available metabolic measurement system (SensorMedics, Vmax, Loma Linda, CA). The metabolic measurement system was calibrated prior to each testing session according to the specifications provided by the manufacturer. The flow sensor was calibrated with a 3.0 liter syringe, while the oxygen (O 2 ) and carbon dioxide (CO 2 ) analyzers were calibrated using reference gases of known concentrations. All pulmonary gas exchange data was averaged over 10 second bins following the completion of the ramp assessment and was used in order to estimate the lactate threshold (LT) and identify the peak oxygen uptake (VO 2pk ). The LT was estimated using the V-slope, end-tidal gases, and ventilatory equivalents as described previously [57, 58]. VO 2pk was defined as the highest O 2 uptake (VO 2 ) value (using the 10 s averaged data) achieved during the maximal ramp exercise test. Heavy Intensity Constant Work Rate Exercise Protocol 45

60 Each subject completed a 20 minute heavy intensity (>LT) square wave exercise bout at a work rate equivalent to 10% above their LT (LT+10%; 157 ± 44.5 W; 54 ± 8% VO 2pk ) combine with a randomized BFR condition on three separate occasions. The BFR conditions used in the present investigation included control (CON, 0 mmhg), low occlusion (LO, 50 mmhg), and high occlusion (HO, ± 6.1 mmhg). The HO condition was equivalent to 130% of the subject's resting systolic blood pressure (130% SBP) and is consistent with occlusion pressures used in previous BFR investigations [15, 36, 42, 53, 101]. The distance from the anterior superior iliac spine (ASIS) to the superior border of the patella was measured and an occlusion cuff (Hokanson, SC5, Bellevue, WA, 6.0 cm width) was positioned 33% of the distance distal to the ASIS, bilaterally. This positioning of the occlusion cuff is similar to the cuff position used in a previous BFR exercise investigation [100]. Surface electromyography (semg) and near-infrared spectroscopy (NIRS) techniques were used to measure the neuromuscular activation and microvascular oxygenation during the heavy intensity cycling exercise bout, respectively. Pulmonary gas exchange was also collected breath-by-breath during the cycling exercise for the determination of oxygen uptake (VO 2 ). Each subject completed a 5 minute cycling warm-up at 20 W followed by a transition in work rate corresponding to LT+10% for 20 minutes. The subject was asked to maintain a pedaling cadence between 80 and 100 rpm during exercise. During the LO and HO conditions the occlusion cuffs were immediately inflated (LO = 50 mmhg, HO = 130%SBP, 164 ± 6 mmhg) at the beginning of exercise using a rapid cuff inflation system (Hokanson, E20 Rapid Cuff Inflator, Bellevue, WA). Following the completion 46

61 of the exercise bout the occlusion cuffs were immediately deflated and the work rate was reduced to 20 W for a 5 minute cool-down period. Subsequent to the cool-down, the subject was transferred from the stationary cycle ergometer to a chair in order to collect the post-exercise arterialized venous blood samples. Blood Sampling Arterialized venous blood was sampled before and after each heavy intensity exercise bout for determination of plasma vascular endothelial growth factor receptor-2 (VEGF-R2) and plasma lactate concentrations. Prior to the exercise bout, the subject rested in a supine position as a percutaneous Teflon catheter (22 gauge, Smiths Medical International Ltd, Rossendale Lancashire, UK) was placed into the dorsal venous plexus of the hand and secured by tape. The blood samples were "arterialized" by warming the forearm and hand with use of a heating pad, as previously described [118, 119]. The arterialized-venous blood was sampled during baseline cycling (20W), immediately post exercise and at 10, 30 and 60 minutes post exercise. All arterialized-venous blood samples were immediately centrifuged and separated (16,100 g, 10 minutes; Microcentrifuge 5415R, Eppendorf, Hauppauge, NY). All plasma samples were extracted and then stored in a -80 C freezer until later analysis. An enzyme-linked immunosorbent assays (ELISA, Human VEGF-R2 ELISA, RayBiotech, Norcross, GA) was used to determine plasma VEGF-R2 concentrations in the arterialized-venous samples at each sample interval. Plasma lactate concentrations ([lactate]) was determined via a blood gas analyzer (Stat Profile phox Plus L, Nova Biomedical, Waltham, MA) at baseline and immediately post exercise. Surface Electromyography (semg) Techniques 47

62 Surface electromyography (semg) was used to examine the neuromuscular activation of the vastus lateralis (VL-RMS) and vastus medialis (VM-RMS) during cycling exercise for each occlusion condition. Prior to placement of the semg electrodes, the skin was shaved and cleansed with an alcohol pad. Double differential semg electrodes (Delsys, Bagnoli 8-Channel System, Boston, MA) with a fixed interelectrode distance of 1 cm were placed on the vastus lateralis and vastus medialis of the left thigh. The vastus lateralis semg electrode was placed at an oblique angle approximately 3-5 cm proximal to the patella just lateral to the midline over the muscle belly [103]. The vastus medialis semg electrode was placed at an oblique angle approximately 2 cm medially from the superior border of the patella within the distal third of the vastus medialis muscle belly [103]. The raw semg signal was collected at a sampling frequency of 2,000 Hz using LabChart 7.0 (ADInstruments, Inc., Colorado Springs, CO). Near-Infrared Spectroscopy (NIRS) Techniques Frequency-domain multi-distance (FDMD) near-infrared spectroscopy (NIRS) was used to continuously monitor absolute deoxy-hemoglobin (deoxy-[hb+mb]) and oxy-hemoglobin (oxy-[hb+mb]) responses. Total hemoglobin concentration ([THC]), an indication of blood volume within the microvasculature, was calculated as the sum of the deoxy-[hb+mb] and oxy-[hb+mb] signals. The NIRS system (Oxiplex TS, ISS, Champaign, IL) was calibrated prior to each data collection session according to the specifications provided by the manufacturer and operated at wavelengths of 690 nm and 830 nm with a sampling frequency of 2 Hz. It has been previously reported that the deoxy-[hb+mb] signal is less sensitive to changes in blood flow perfusion (reflected by 48

63 Δ[THC]) compared to the oxy-[hb+mb] signal [104] and that the deoxy-[hb+mb] response is an acceptable estimation of oxygen extraction [105, 106]. Although the microvascular deoxygenation response within the vastus lateralis has been shown to be non-uniform throughout the muscle during fatiguing exercise, the greatest microvascular deoxygenation response has been observed at the middle region of the vastus lateralis during knee extension exercise compared to the distal regions [107]. Therefore, the NIRS sensor was positioned midway between the ASIS and the superior border of the patella over the muscle belly of the vastus lateralis in the present investigation. Care was taken to place the NIRS sensor in the same position for each exercise condition. Prior to placement of the NIRS sensor the skin was shaved and cleansed with an alcohol pad. The NIRS sensor was covered with a dark black cloth, in order to prevent stray visible light sources from affecting the data acquisition, and secured with straps placed over the vastus lateralis muscle. Data Processing The raw semg data were amplified by a gain of 1,000 (Bagnoli 8-Channel System, Delsys, Boston, MA), digitally filtered using a band-pass filter (10Hz-500Hz) and smoothed using a 50 millisecond root means squared (RMS) window. All neuromuscular activation, microvascular oxygenation and oxygen uptake data was averaged into 10 second bins. Then neuromuscular activation, microvascular oxygenation and oxygen uptake measurements were analyzed in 60 second bins at 1, 5, 10, 15, and 19 minutes into the heavy intensity (>LT) exercise bout. All semg data was normalized as a percentage of the baseline neuromuscular activation (%BL) that occurred during the last 30 seconds of the warm-up cycling (20W) prior to the heavy intensity 49

64 work rate transition, similar as previously described [120]. All NIRS data was normalized as a percentage of the baseline microvascular oxygenation (%BL) that occurred during the last 30 seconds of the warm-up cycling (20 W) prior to the heavy intensity work rate transition. Statistical Analysis A two-way (occlusion condition x time) analysis of variance (ANOVA) with repeated measures was used to identify differences in the dependent variables (plasma VEGF-R2 concentrations, [lactate], neuromuscular activation (RMS), deoxy-[hb+mb], [THC], and oxygen uptake). Tukey's post-hoc analysis was used in order to find significance when appropriate. Statistical significance was set at a priori of p All values are expressed as the group mean ± standard deviation (SD) unless otherwise stated. All statistical analyses was completed using Sigma Stat software (Sigma Stat 3.5, Systat Software, San Jose, CA) Results Surface Electromyography (semg) Results HO (202.9 ± 50.2%BL) resulted in a lower neuromuscular activation of the vastus lateralis (VL-RMS) compared to CON (256.2 ± 43.2 %BL) during the last minute of the heavy intensity exercise bout (p = 0.034). The last minute (256.2 ± 43.2%BL) of the heavy intensity exercise bout elicited a greater neuromuscular activation of the VL-RMS compared to the first minute (196.4 ± 37.4%BL) of the heavy intensity exercise bout during the CON condition (p = 0.021). There were no significant differences observed in neuromuscular activation at any of the time points examined within or between the occlusion conditions for either the VL-RMS or vastus medialis (VM-RMS). 50

65 Near-Infrared Spectroscopy (NIRS) Results Due to technical difficulties with the computer, the microvascular oxygenation response for one subject could not be retrieved and therefore the NIRS responses are for seven of the subjects. The HO condition resulted in greater deoxy-[hb+mb] compared to CON at 1 minute (HO = ± 14.7%BL; CON = ± 19.9%BL; p = 0.010), 5 minutes (HO = ± 14.5%BL; CON = ± 16.8%BL; p = 0.007), and the last minute of exercise (HO = ± 13.0%BL; CON = ± 15.7%BL; p = 0.041). HO also resulted in greater deoxy-[hb+mb] compared to LO at 5 minutes (HO = ± 14.5%BL; LO = ± 16.8%BL; p = 0.030) and the last minute of exercise (HO = ± 13.0%BL; LO = ± 19.5%BL; p = 0.036). During CON, deoxy-[hb+mb] was greater during the end of exercise (10, 15 and 19 minutes) compared to the beginning of exercise (1 and 5 minutes). Additionally, deoxy-[hb+mb] was greater at 5 minutes compared to the first minute of the CON exercise bout. LO exercise resulted in greater deoxy-[hb+mb] during the end of exercise (10, 15 and 19 minutes) compared to the first minute of exercise. Furthermore, HO exercise elicited greater deoxy-[hb+mb] during the end of exercise (5, 10, 15 and 19 minutes) compared to the first minute of exercise. In addition, HO resulted in higher [THC] compared to CON at 5 minutes (HO = ± 7.6%BL; CON = ± 2.6%BL; p = 0.010), 10 minutes (HO = ± 9.8%BL; CON = ± 3.5%BL; p = 0.013), and 15 minutes (HO = ± 7.1%BL; CON = ± 5.0%BL; p = 0.033) of the heavy intensity bout of exercise. HO also resulted in greater [THC] at 10 minutes compared to the first minute of exercise. Oxygen Uptake (VO 2 ) Results 51

66 There was no significant differences observed in VO 2 during the heavy intensity exercise between any of the BFR conditions examined (CON, LO, HO) within the exercising time points (1 min, 5 min, 10 min, 15 min, 19 min). However, VO 2 was greater during the following time points (5 min, 10 min, 15 min, 19 min) compared to 1 minute within all exercise conditions. Arterialized Venous Blood Sampling Results A significant main effect for occlusion condition was detected for VEGF-R2 as HO (78.7 ± 1.8 pg/ml) was higher compared to CON (CON = 77.7 ± 1.0 pg/ml; p = 0.030). However, there were no significant differences detected in VEGF-R2 subsequent to the heavy intensity exercise bout between any of the exercising conditions (CON, LO, HO) within the timed blood samples (Pre, 10 Post, 30 Post, 60 Post). As expected, a significant main effect for time was detected for plasma [lactate] as immediate post exercise (10.1 ± 4.3 mmol/l) was higher compared to baseline (BSL = 2.8 ± 1.2 mmol/l; p = 0.002). However, no significant difference was observed for plasma [lactate] between any of the occlusion conditions (CON, LO, HO) within the timed blood samples (baseline and immediately post exercise) Discussion In contrast to our first hypothesis and previous BFR investigations [16-19], the neuromuscular activation during heavy intensity (VT+10%) cycling exercise was not different, or even lower during the last minute of exercise, during HO compared to both CON and LO within the vastus lateralis (Figure 4.1). It has been previously suggested that hypoxia causes a decline in neuromuscular activation (root means squared, RMS) following a sustained sub-maximal isometric contraction [121]. The repetitive cyclic 52

67 contractions performed at high contraction velocities ( rpm) during the cycling exercise potentially over time could have elicited a similar response to the muscle thus potentially explaining the declined neuromuscular activation found within the vastus lateralis towards the end of exercise (19 min). However, this relationship between neuromuscular activation and the effects of hypoxia from BFR techniques during cardiovascular exercise should be investigated further. It is important to note, that the increase in neuromuscular activation reported previously [16-19] have been observed using resistance exercise models, in which the contraction duration and contraction velocity differ from cycling exercise. The results of the present investigation suggest that exercise mode (i.e., resistance exercise, cardiovascular exercise) may influence the effects of BFR on neuromuscular activation. In addition, the effects of varied contraction velocities on neuromuscular activation during BFR exercise, during both resistance and aerobic exercise modes, warrants further investigation. However, supporting our first hypothesis, HO elicited a greater deoxy-[hb+mb] compared to CON and LO during the beginning and end of the heavy intensity cycling exercise (Figure 4.2). The deoxy-[hb+mb] signal provides an estimate of the oxygen extraction (A-VO 2diff ) within the field of interrogation [114] and has been demonstrated as being insensitive to blood volume changes [104, 122]. The increased deoxy-[hb+mb] during HO exercise supports the previous finding of a greater oxygen extraction during ischemic cycling exercise [81]. However, similar to a previous BFR investigation [45], the present investigation found no difference in exercising VO 2 between any of the exercising conditions (Figure 4.4), despite the greater deoxy-[hb+mb] signal during HO exercise. According to Fick's equation, this finding could suggest that the perturbation 53

68 caused by HO may have affected cardiac output in some way, however, this was managed by the adjustment of an increased oxygen extraction (i.e., increased deoxy- [Hb+Mb]). The author's of the present investigation can only speculate the effect that BFR cardiovascular exercise has on cardiac output as it was not directly measured during the present investigation and deoxy-[hb+mb] is only used as a surrogate for oxygenation extraction [114]. This speculation differs from a previous BFR investigation [45], that observed no difference in VO 2 or cardiac output between sub-maximal BFR and control cycling exercise when stroke volume and heart rate were measured non-invasively. Further evidence is required to determine the hemodynamic effects of BFR cycling exercise. However, the higher deoxy-[hb+mb] observed during HO exercise, could suggest the presence of a greater hypoxic intramuscular environment [105, 106], thus providing support for the lowered neuromuscular activation demonstrated during HO exercise. Increases in [THC], a measurement of blood volume within the area of interrogation [114], was observed during HO compared to CON during the heavy intensity cycling exercise (Figure 4.3). The elevated [THC] could be explained by the presence of venous pooling within the exercising musculature, as a reduction in venous return has previously been associated with BFR exercise [53, 110]. It has been proposed that the venous pooling related to BFR exercise could promote "cellular swelling", a potential mechanistic link to the increase in hypertrophy following BFR training [80]. However, the authors of the present investigation caution this immediate explanation as the increased [THC] during exercise could also be explained by recruitment of adjacent vessels within the area of interrogation during exercise. Furthermore, [THC] did not 54

69 differ between CON and LO exercising conditions at any point during the exercise bout, thus suggesting that the low external occlusion pressure (LO, 50 mmhg) applied may not be a sufficient stimulus if venous pooling is a required BFR stimulus for training adaptations to occur. Contrary to our second hypothesis, plasma VEGF-R2 concentrations were not altered from baseline values when compared between the exercise conditions within time (Table 4.2), although a significant main effect was detected as HO elicited higher plasma VEGF-R2 concentrations when compared to CON. It is possible that plasma VEGF-R2 continued to rise after the blood sample collections for the present investigation as it has been suggested that peak plasma VEGF-R2 concentration occurs approximately 2 to 4 hours post exercise [123] and samples were obtained at 10, 30, and 60 minutes post exercise during the present investigation. However, significant increases in plasma VEGF-R2 have been detected minutes post exercise [123]. Therefore, it could be suggested that a detectable increase in plasma VEGF-R2 should have been discovered during the blood sample collections of the present investigation, if an increase were present, even if the plasma VEGF had not reached peak concentrations subsequent to exercise. Despite the possible sample timing differences, previously, low intensity (20% one repetition maximum, 1RM) BFR knee extension exercise has shown significant increases in plasma VEGF-R2 during exercise and 10 and 30 minutes post exercise [53]. However, similar to the present results, no change in plasma VEGF-R2 has also been demonstrated following 40% 1RM BFR knee extension exercise at 4 and 24 hours post exercise [43]. Despite no difference in plasma VEGF-R2 following BFR knee extension 55

70 exercise, significant increases in skeletal muscle expression of VEGF-R2 was observed 4 hours subsequent to BFR knee extension exercise via muscle biopsy samples [43]. Two factors that could potentially explain the results of the previous and present investigations findings include 1) exercise modes and 2) fatigue status of the subject. The exercise mode could have affected the plasma VEGF-R2 response as both of the previous investigations [43, 53] that have demonstrated increases in plasma VEGF-R2 have used a resistance exercise model during BFR exercise rather than a cardiovascular exercise model. In addition, BFR resistance exercise has previously been reported to increase the neuromuscular activation of the exercising muscles [16-19], which was not observed during BFR cardiovascular exercise during the present investigation (Graph 4.1). Significant elevations in VEGF have been detected (in animal models) subsequent to increased neuromuscular activation [78, 124]. Although only speculation, it could be possible that the increased neuromuscular activation observed during BFR resistance exercise could be associated with the increased VEGF-R2 and neither response (neuromuscular activation, VEGF-R2) are associated with BFR cycling exercise. Furthermore, the fatigue status of the subject could potentially affect the plasma VEGF-R2 response as the subjects of the previous investigations [43, 53] were asked to perform exercise to volitional fatigue whereas the subjects of the present investigation only completed 20 minutes of cycling exercise and where not fatigued at the end of the cycling exercise bout. In support of the fatigue response, no significant increases in plasma lactate were observed between any of the cycling conditions immediately post exercise (Table 4.3), suggesting that the metabolic stress of each cycling exercise condition were similar. 56

71 4.5 - Conclusion The primary objective of the present investigation was to determine the acute physiological responses to BFR cycling exercise in an attempt to provide a mechanistic link to the previously observed increases in VO 2pk following BFR training [14, 15, 21]. According to the present results, VO 2 does not appear to be affected by heavy intensity BFR cycling exercise. However, the perturbations caused by the application of BFR (HO) within the present investigation was managed by a more localized response as oxygen extraction increased (i.e., increased deoxy-[hb+mb]). Furthermore, no changes were detected in neuromuscular activation between exercising conditions with the exception for end exercise where a significant decline in neuromuscular activation was observed possibly due to limited O 2 availability. Finally, conflicting with previous BFR resistance exercise investigations, the present investigation demonstrated that an acute bout of heavy intensity cycling exercise did not affect the subsequent plasma VEGF-R2 response although increases in plasma [lactate] where observed. This finding provides evidence that the plasma VEGF-R2 response subsequent to the performance of BFR exercise may be more dependent upon exercise mode (cycling exercise, resistance exercise). 57

72 Figure 4-1 Neuromuscular activation of the vastus lateralis was significantly lower for high occlusion cycling (HO, green bar) compared to control cycling (CON, black bar) during the last minute of the heavy cycling exercise. *Significantly different from CON (p < 0.05). +Significantly different from LO (p < 0.05). 58

73 Figure 4-2 High occlusion cycling (HO, green bar) elicited greater microvascular deoxygenation (Deoxy-[Hb+Mb]) compared to control cycling (CON, black bar) at one, five, and nineteen minutes of the heavy cycling exercise bout. HO cycling resulted in greater deoxy-[hb+mb] compared to low occlusion cycling (LO, red bar) at five and nineteen minutes of the heavy cycling exercise bout. *Significantly different from CON (p < 0.05). +Significantly different from LO (p < 0.05). 59

74 Figure 4-3 High occlusion cycling (HO, green bar) resulted in significantly greater total hemoglobin concentration ([THC]) compared to control cycling (CON, black bar) at five, ten, and fifteen minutes of the heavy cycling exercise bout. *Significantly different from CON (p < 0.05). +Significantly different from LO (p < 0.05). 60