Metabolic Response to Provision of Mixed Protein-Carbohydrate Supplementation During Endurance Exercise

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1 ORIGINAL 384 / Miller et al. RESEARCH International Journal of Sport Nutrition and Exercise Metabolism, 2002, 12, Human Kinetics Publishers, Inc. Metabolic Response to Provision of Mixed Protein-Carbohydrate Supplementation During Endurance Exercise Sharon L. Miller, Carl M. Maresh, Lawrence E. Armstrong, Cara B. Ebbeling, Shannon Lennon, and Nancy R. Rodriguez The interaction of substrates and hormones in response to ingestion of intact proteins during endurance exercise is unknown. This study characterized substrate and hormone responses to supplementation during endurance exercise. Nine male runners participated in 3 trials in which a non-fat (MILK), carbohydrate (CHO), or placebo (PLA) drink was consumed during a 2-hour treadmill run at 65% V O 2max. Circulating levels of insulin, glucagon, epinephrine, norepinephrine, growth hormone, testosterone, and cortisol were measured. Plasma substrates included glucose, lactate, free fatty acids, and select amino acids. Except for insulin and cortisol, hormones increased with exercise. While postexercise insulin concentrations declined similarly in all 3 trials, the glucagon increase was greatest following MILK consumption. CHO blunted the postexercise increase in growth hormone compared to levels in MILK. Free fatty acids and plasma amino acids also were responsive to nutritional supplementation with both CHO and MILK attenuating the rise in free fatty acids compared to the increase observed in PLA. Correspondingly, respiratory exchange ratio increased during CHO. Essential amino acids increased significantly only after MILK and were either unchanged or decreased in CHO. PLA was characterized by a decrease in branched-chain amino acid concentrations. Modest nutritional supplementation in this study altered the endocrine response as well as substrate availability and utilization following and during an endurance run, respectively. Key Words: substrates, hormones, runners, milk, amino acids, protein Introduction The role of protein in the endurance athlete s diet is a current topic of interest in sports nutrition. Several popular diets focus on protein as the key component in a The authors are with the Department of Nutritional Sciences at the University of Connecticut, Storrs, CT In addition, C.M. Maresh, L.E. Armstrong, and N.R. Rodriguez are with the Department of Kinesiology at the University of Connecticut, and Maresh and Armstrong are with the Department of Physiology and Neurobiology at the University of Connecticut. 384

2 Protein-Carbohydrate and Endurance Exercise / 385 nutritional approach to achieving optimal body composition and to promote recovery during training (12, 27). Proteins or amino acids also are key ingredients in many sports drinks and bars designed to improve muscle anabolism. Some supplements are promoted as aids to help the athlete attain the balance of macronutrients purported to benefit muscle growth and performance. Others promise to enhance muscle hypertrophy in response to exercise by supplying on demand the necessary building blocks for protein synthesis. While many resistance trained athletes use proteinbased supplements, the endurance athlete typically relies upon carbohydrate supplements. Past endurance exercise studies have focused on carbohydrate supplementation and utilization given that sufficient exogenous carbohydrate modifies the fuel mix relied upon during prolonged exercise and typically favors performance (18). Carbohydrate ingestion during exercise has been shown to maintain blood glucose levels (17), increase carbohydrate oxidation (28, 34), decrease free fatty acid concentrations (23), and decrease leucine oxidation (8). Few studies have explored the integrated response of substrates and hormones when intact protein is consumed during endurance exercise. Studies that have compared traditional carbohydrate supplements to protein or amino acid supplements have sought to demonstrate that protein-based supplements establish a less catabolic environment by (a) providing substrate directly, thus minimizing utilization of endogenous stores of glycogen and protein (2, 7), and (b) enhancing the balance of anabolic to catabolic hormones that is, increasing testosterone while decreasing cortisol (5, 6, 31). The efficacy of branched-chain amino acid (BCAA) mixtures in improving performance has been investigated on two levels, first via amino acid regulation of muscle protein metabolism (14, 21) and, second, with regard to the central fatigue hypothesis (9). Recent investigations of this nature (3, 10) have addressed the potential for mixed supplements that provide both amino acids and carbohydrate to elicit the most favorable response by satisfying substrate needs in addition to altering the neurotransmitter profile to deter central fatigue. Studies have not been done, however, to evaluate whether a protein-containing supplement consumed during endurance exercise impacts the endocrine and substrate environment during recovery. While provision of protein during endurance exercise is not a common consideration, it is important to assess the metabolic response to this type of supplement when considering different aspects of protein utilization during recovery. The present study was designed to characterize the metabolic outcome of an endurance run during which fat-free milk, representing a relatively low-energy (170 kcals) mixed carbohydrate- protein beverage, was provided to trained athletes. Preand post-exercise plasma substrate and hormone assessments were complemented by respiratory exchange ratio (RER) measures throughout exercise. The endocrine and substrate profile elicited by consumption of skim milk was compared to that observed in response to a traditional carbohydrate drink. In addition, the metabolic outcome of supplemented exercise was contrasted to observations made following a non-supplemented bout. We hypothesized that provision of a mixed carbohydrateprotein beverage throughout a prolonged run of moderate intensity would elicit a metabolic profile distinct from that observed when a carbohydrate or non-nutritive supplement was consumed with regard to substrate utilization during exercise and the hormonal and metabolic environment post exercise.

3 386 / Miller et al. Subjects Methods Following project approval by the Institutional Review Board for Studies Involving Human Subjects at the University of Connecticut, 9 male endurance athletes were recruited from the community to participate in the study. Volunteers provided written, informed consent and completed a medical health history questionnaire. All subjects were determined to be in good health and free of metabolic disorders. A minimum running mileage of 35 miles per week for at least 3 months was used as criteria for inclusion in the study. Physical characteristics of the subjects are provided in Table 1. Study Protocol Each subject participated in three randomly ordered trials in which either a mixed carbohydrate-protein supplement (MILK), a carbohydrate supplement (CHO), or a non-nutritive supplement (PLA) was administered during the endurance bout. Trials were separated by at least 7 but not more than 10 days. Prior to initiation of the trials, each athlete reported to the laboratory for collection of baseline measures, which included assessment of maximal oxygen consumption (V O 2max ) and determination of body composition by hydrostatic weighing. Subjects also performed a running trial designed to elicit the treadmill speed corresponding to 65% of the individual s predetermined V O 2max that would be employed during subsequent exercise trials. All exercise trials were performed with the treadmill flat (no grade). Vigorous physical activity was restricted on the day preceding a trial, as was consumption of alcohol or caffeine. Three-day food records were obtained from subjects following enrollment in the study and prior to each trial. Average energy intake and macronutrient composition of the diets were determined by a registered dietitian using Nutritionist IV software (N 2 Computing, Salem, OR).The exercise protocol is depicted in Figure 1. Trials were conducted in the morning following an overnight fast and involved a 2-hour treadmill run at 65% of V O 2max. Prior to exercise a 19 G Teflon catheter (Jelco, Critikon, Tampa, FL) was placed into a superficial forearm vein for pre- and post-exercise blood sampling. Table 1 Subject Characteristics Variable Value Age (years) 23.2 ± 0.6 Height (cm) ± 1.4 Weight (kg) 72.3 ± 1.2 Body fat (%) 13.9 ± 0.8 V O 2max ml kg 1 min ± 0.5 Note. Values are means ± SE of 9 subjects.

4 Protein-Carbohydrate and Endurance Exercise / 387 Figure 1 Exercise protocol. Subjects drank the pre-measured 200-ml volume of supplement while straddling the treadmill at 20, 40, 60, and 80 min of exercise. The carbohydrate drink consisted of 45 g of dextrose (table sugar) mixed with 835 ml of bottled water and non-sweetened flavoring (Kool-Aid, Kraft, Inc., White Plains, NY). The protein supplement consisted of 480 ml of skim milk diluted with 355 ml of bottled water. Flavoring and aspartame (Equal, The Nutrasweet Company, Deerfield, IL) were added to the drink in an effort to mask the nature of its composition and to increase palatability. The MILK supplement contained 17 g of protein and 27 g of carbohydrate. The placebo drink was prepared using bottled water, non-sweetened flavoring, and aspartame. The carbohydrate and protein drinks were isocaloric, providing ~170 kcals in total. Water was available to subjects throughout the endurance trials, and temperature of the laboratory was maintained between ºC during all studies. Indirect calorimetry readings were taken at 15 min of exercise and 5 min after each portion of supplement was consumed to monitor V O 2 and to record RER. Analytical Methods Hormones. All hormones were assayed in duplicate. Norepinephrine (NE) and epinephrine (EPI) were analyzed by high-pressure liquid chromatography (HPLC) with electrochemical detection following extraction from plasma with an ion exchange column (Chromsystems, Munich, Germany). Plasma concentrations of insulin, glucagon, cortisol, testosterone, and growth hormone were measured by radioimmunoassay (Coat-a-Count Insulin, TKIN2, Coat-a-Count Cortisol, TKCO2, Double Antibody Glucagon, KGND1, Double Antibody Human Growth Hormone, KGHD2 Diagnostic Products Corp., Los Angeles, CA). The ratio between testosterone and cortisol was calculated and interpreted to reflect anabolic/catabolic steroid balance (1). Substrates. Blood samples were processed and analyzed in duplicate for determination of pre- and post-exercise glucose and lactate concentrations. Glucose was analyzed by an automated glucose oxidase method (YSI Model 2300, Yellow Springs, OH) and lactate concentrations were measured by enzymatic assay simultaneously. If samples could not be analyzed on the day of the study, they were frozen at 80 ºC

5 388 / Miller et al. and analyzed at a later date. Plasma concentrations of total non-esterified free fatty acids (FFA) were measured using an enzymatic, colorimetric assay (NEFA C ACS- ACOD Method, Wako Chemicals, Richmond, VA). Plasma amino acids were quantified by reverse phase HPLC analysis using the Pico-Tag method (Waters Chromatography Division, Milford, MA). Statistical Analysis. Data are presented as means ± standard error of the mean (SEM). Treatment means were calculated for all variables. Normally distributed means were compared using analysis of variance (ANOVA) and paired Student s t- test. When significant differences were indicated by ANOVA, Tukey s post hoc analysis was applied to identify specific differences. Respiratory exchange ratio data were analyzed using repeated measures ANOVA. The level for statistical significance was set at Statistical analyses were conducted using SAS (Cary, NC). Results Average V O 2 during exercise was 2.8 ± 0.06 L min 1, which represented 67 ± 0.8% of predetermined maximal oxygen consumption. Athletes ran ~20 ± 3 km during the 2-hour run. Plasma volume changed (p <.05) by 4.8 ± 2, 6.7 ± 3, and 3.3 ± 2% in PLA, CHO, and MILK trials, respectively. Plasma concentration values were not corrected for these changes. Three-day diet records showed that energy intake and macronutrient composition of the subjects diets prior to each trial did not differ within or between subjects. Protein intake exceeded the RDA (1.88 ±.7 g kg 1 day 1 ) but approximated values reported for endurance athletes (24). Carbohydrate intake preceding each study was adequate, providing 6.2 ± 0.5 g kg 1 day 1. Hormones All hormones assayed changed significantly in response to exercise, but only growth hormone and glucagon were influenced by nutritional supplementation (Table 2). Growth hormone concentration increased significantly (p <.05) post exercise in all trials. Carbohydrate supplementation suppressed the increase in GH such that postexercise values in the CHO trial were significantly lower than those observed following MILK (p <.05) and tended to be lower than levels following PLA (p =.07). Glucagon concentrations were elevated post exercise (p <.05) with changes in MILK trial being greater (p <.05) than those observed in the PLA trial. In contrast, insulin concentrations decreased (p <.05) following exercise but did not differ between trials. As with the other counter-regulatory hormones, plasma norepinephrine and epinephrine increased (p <.05) following exercise. However, the changes in concentration were not significantly different between trials. Plasma cortisol values decreased (p <.05) to the same extent for all three conditions pre to post exercise. Free testosterone concentrations increased (p <.05) in response to exercise but, like cortisol, did not respond to nutritional supplementation. Therefore, the testosterone:cortisol ratio increased (p <.05) similarly in all trials. Substrates Plasma lactate, glucose, and free fatty acid values (Table 2) increased in response to exercise, whereas plasma amino acids changed in different directions depending upon the supplement provided. Both plasma glucose and lactate tended to be higher

6 Protein-Carbohydrate and Endurance Exercise / 389 before the start of exercise in the CHO trial compared to MILK and PLA. These unexplained differences were not sustained after exercise when glucose and lactate values increased (p <.01) in all trials. Despite the different pre-exercise values for glucose and lactate in the CHO trial, neither the change in concentrations nor the absolute post-exercise values differed between treatments. Free fatty acids were elevated following exercise (p <.05) and responded to nutritional supplementation as evidenced by significantly lower levels in CHO and MILK compared to PLA (p <.05). Plasma concentrations of select amino acids and the branched chain amino acids (BCAA) are presented in Table 3. Change scores for amino acid concentrations pre to post exercise are represented in Figure 2. In general, MILK supplemented exercise yielded increases in amino acid concentrations relative to pre-exercise concentrations (p <.05), while CHO Figure 2 Select amino acid change scores (change pre- to post-exercise). Values are means ± SEM for 9 subjects. a Significantly different from pre-exercise value (p <.05). b Significantly different from 0 (p <.001).

7 390 / Miller et al. Table 2 Plasma Hormones and Substrates Pre and Post Exercise PLA CHO MILK Variable Pre Post Pre Post Pre Post Hormones Insulin ( U ml 1 ) 7.3 ± ± 0.6 * 7.7 ± ± 0.7 * 7.6 ± ± 1.0 * Glucagon (pg ml 1 ) 82.5 ± ± 6.0 * 81.9 ± ± 8.0 * 87.8 ± ± 6.0 Growth hormone (ng ml 1 ) 2.5 ± ± 1.2 * 0.9 ± ± 2.0 * 0.32 ± ± 2.1 * Norepinephrine (pg ml 1 ) ± ± 49 * ± ± 215 * ± ± 170 * Epinephrine (pg ml 1 ) 46.5 ± ± 36 * 34.7 ± ± 25 * 35 ± ± 42 * Testosterone (nmol L 1 ) 20.8 ± ± 2.7 * 24.4 ± ± 2.5 * 23.9 ± ± 2.5 * Cortisol (nmol L 1 ) ± ± 73 * ± ± 57 * ± ± 77 * Substrates Glucose (mg dl 1 ) 86.7 ± ± 2.6 * 89.4 ± ± 3.1 * 86.5 ± ± 2.1 * Lactate (nmol L 1 ) 0.84 ± ±.11 * 1.11 ± ±.15 * 1.03 ± ±.18 * Free fatty acids (meq L 1 ) 0.38 ± ±.1 * 0.39 ± ±.08 * 0.38 ± ±.08 * *, ** * Significantly different from Pre, p <.05; ** significantly greater than PLA, p <.05; significantly greater than CHO, p <.05; significantly greater than CHO and MILK, p <.05.

8 Protein-Carbohydrate and Endurance Exercise / 391 Table 3 Plasma Amino Acid Concentrations Pre and Post Exercise for Each Treatment PLA CHO MILK Amino acids ( mol/l Pre Post Pre Post Pre Post Alanine 315 ± ± ± ± ± ± 18 Arginine 60 ± 3 64 ± 5 64 ± 5 55 ± 5 60 ± 5 71 ± 6 b Glutamate 28 ± 6 47 ± 9 47 ± 9 56 ± 7 35 ± 7 55 ± 9 Glycine 188 ± ± ± ± ± ± 10 Leucine 121 ± ± ± ± ± ± 9 c Isoleucine 58 ± 4 48 ± 4 57 ± 6 43 ± 4 54 ± 5 56 ± 5 c Lysine 119 ± ± ± ± ± ± 7 c Methionine 23 ± 2 27 ± 2 24 ± 3 23 ± 2 20 ± 2 29 ± 2 c Phenylalanine 50 ± 3 61 ± 4 d 51 ± 6 48 ± 3 46 ± 3 57 ± 2 Threonine 88 ± 6 84 ± 7 97 ± 9 77 ± 8 72 ± 8 a 82 ± 7 Tryptophan 47 ± 3 50 ± 5 48 ± 4 46 ± 4 44 ± 3 54 ± 4 Tyrosine 55 ± 3 65 ± 5 58 ± 5 54 ± 4 48 ± 4 75 ± 6 d Valine 199 ± ± ± ± ± ± 15 Note. Values are means ± SE for 9 subjects. a Less than CHO Pre, p <.05. b Greater than PLA Post, p <.05. c Greater than CHO Post, p <.05. d Greater than CHO Post, p <.05.

9 392 / Miller et al. supplemented exercise was characterized by decreases in amino acid concentrations (p <.05). In both PLA and CHO, BCAAs decreased significantly from pre to post exercise (p <.05), while pre-exercise levels were maintained with MILK. Consequently, the pre- to post-exercise change in BCAAs was positive in MILK supplemented exercise relative to negative changes in PLA and CHO. Glutamate concentrations increased in all trials, but the change was significant only in PLA and MILK trials. Respiratory Exchange Ratio (RER) Respiratory exchange ratio (RER) decreased significantly (p <.05) for PLA compared to CHO during the run. Although a time by treatment interaction was not significant (p =.08), RER was significantly higher (p <.05) in the CHO trial relative to PLA at 65 and 85 min of exercise (Figure 3). Discussion This study characterized the metabolic response to consumption of a traditional carbohydrate (CHO) and rather non-traditional mixed carbohydrate-protein (fatfree MILK) supplement during an endurance run. Low-energy supplementation, in general, did not profoundly affect the endocrine environment during prolonged treadmill exercise, since insulin decreased and counter-regulatory hormones increased to a similar degree in all trials. Accordingly, post-exercise testosterone and cortisol concentrations were not responsive to acute nutritional supplementation at this dosage. Others have observed an influence of diet as well as acute supplementation on cortisol and testosterone during recovery from strength and endurance training (5, 6, 20, 31). In a study by Carli et al. (5), the addition of branched-chain amino Figure 3 Respiratory exchange ratio (RER) in early exercise (15 min) and 5 min following provision of supplement.

10 Protein-Carbohydrate and Endurance Exercise / 393 acids (BCAA) to a mixed carbohydrate-protein supplement ingested prior to a running event did not affect cortisol values immediately post exercise. However, by 1 hour after exercise, cortisol concentrations were lower in the BCAA trial compared to recovery levels observed after exercise without amino acid supplementation. Unfortunately, a carbohydrate trial to distinguish between effects of the protein in the supplement and the BCAA was not included in the study design. It is conceivable that nutrient-related changes in the testosterone:cortisol ratio might have occurred relatively late in recovery from supplemented endurance exercise in this study. Delayed effects of supplementation on hormones have been reported in other studies. Chandler and associates (6) noted increased testosterone clearance as well as higher growth hormone levels 5 6 hours after carbohydrate, protein, or mixed supplements were consumed immediately following weight-training exercise. Samples taken immediately post-exercise in the present investigation showed that supplementation did affect GH levels such that CHO inhibited the increase that was observed in MILK and PLA trials. However, no supplement-related differences in testosterone were found post exercise, and it remains speculative whether supplementation may have influenced hormone levels hours after completion of exercise. With regard to plasma insulin and glucagon, neither CHO or MILK prevented the respective decrease and increase that typically follow endurance exercise. Two factors that likely minimized the influence of supplementation on these hormones were the marginal amounts of nutrients provided and ingestion of supplements during, as opposed to before, exercise. While carbohydrate given prior to or following exercise has been shown to increase insulin (23), fewer studies have demonstrated an insulin response to glucose feeding during exercise (16, 28, 34). Given that the carbohydrate load provided in the CHO trial did not increase insulin levels, it is not surprising that insulin concentrations were unaffected by the mixed supplement, which provided a slightly lower amount of carbohydrate. The dose and schedule of supplement provision were chosen in part to prevent large shifts in the physiological steady-state during exercise as well as to avoid gastrointestinal distress. Varying the type of carbohydrate provided and including other components such as protein or fiber (19) can slow nutrient assimilation and moderate the hormonal response to a supplement. The supplementation protocol in this study influenced the endocrine response minimally, although inclusion of protein modified some parameters. While the insulin decline was similar in all three treatments, the magnitude of glucagon increase was greatest with MILK supplementation. Dietary amino acids provided by MILK could have signaled a glucagon response. In resting individuals, carbohydrate consumption increases insulin and decreases glucagon, while parallel increases in insulin and glucagon have been observed with protein feeding (33). The possibility exists that the observed increase in glucagon with protein may be related to observed changes in the catecholamines, since epinephrine has been shown to stimulate glucagon release (11). Although the magnitude of difference in epinephrine concentrations between treatments was not statistically different, protein supplementation elicited approximately a six-fold increase in epinephrine compared to three- and four-fold increases in the hormone following PLA and CHO trials, respectively. Both epinephrine and glucagon act to promote fat oxidation. Epinephrine does so by raising free fatty acids and glucagon while also suppressing whole body proteolysis (15), whereas glucagon stimulates lipolysis and raises free fatty

11 394 / Miller et al. acid concentrations (11). Protein supplementation during exercise, therefore, could potentially elicit a different pattern of substrate utilization after exercise than carbohydrate supplementation. In this study, plasma substrate responses typified those associated with prolonged submaximal exercise, although supplementation did influence the degree of some of these changes. Nutritional supplementation in this study elicited changes in plasma free fatty acids and amino acids, but did not affect glucose levels. Plasma glucose increased to a similar level by completion of exercise in all trials as did plasma lactate with treatment having no effect on post-exercise concentrations. The fact that supplementary CHO was not associated with higher plasma glucose levels suggests that carbohydrate may have been utilized to a greater extent in this trial compared to MILK and PLA. An increase in carbohydrate oxidation with CHO supplementation in this study is supported by the higher respiratory exchange ratio (RER) with CHO supplemented exercise. RER decreased significantly over time in non-supplemented and MILK-supplemented exercise, indicating greater reliance on lipid or non-carbohydrate substrate during later stages of exercise. CHO prohibited the decrease in RER over time and sustained a significantly higher RER throughout the last hour of exercise compared to the PLA trial. By late exercise, the observed RER value with MILK of 0.90 was higher than the calculated RQ value of 0.82 for protein (13) as would be expected, since the milk provided lactose as well as protein. In addition, endogenous fuel stores were likely being utilized at this time. Other studies consistently show accelerated leucine oxidation during exercise and even at rest when protein or amino acids are provided in surplus in the diet or as a supplement (3, 13, 30). Without knowledge of nitrogen disposal, however, it is not possible to determine protein contribution to the fuel mix for these athletes. Both CHO and MILK consumption during exercise suppressed FFA concentrations relative to the value observed in the PLA trial. Consistent with other reports (7, 23), free fatty acids were mobilized and apparently utilized more when no nutritional supplement was given during exercise. Conversely, provision of CHO appeared to promote carbohydrate utilization as fuel. In total, these findings show that fuel utilization during exercise trials involving the same constant duration and intensity responded to substrate availability. Reported values of plasma amino acids following endurance exercise vary but, in general, branched-chain amino acid concentrations decline, while some nonessential amino acids (alanine, glutamine) increase in the plasma (26). In the current study, MILK supplementation prevented the decrease in BCAAs observed in PLA and CHO and significantly increased other essential amino acids. In contrast, CHO supplementation was marked by a general downward change in concentration following exercise. Supplementation with BCAAs consistently increases plasma concentrations of isoleucine, leucine, and valine (10, 21), and most studies have focused on performance improvement via a decrease in TRP:BCAA and consequent deferral of central fatigue. Fewer investigations address the potential for increased amino acid availability to promote muscle protein synthesis (14, 21). A number of studies have assessed the hormonal response to protein-based supplements (5 7). Many infer enhanced muscle anabolism from increased concentrations of anabolic hormones, but no studies have directly assessed muscle protein metabolism in relation to nutrient-supplemented endurance exercise. A recent study (25) found improved muscle protein anabolism following resistance exercise when a mixed amino

12 Protein-Carbohydrate and Endurance Exercise / 395 acid carbohydrate supplement was provided during recovery. A follow up to that study showed even greater delivery and uptake of amino acids by the muscle when the drink was provided before the exercise bout (29). One might speculate that a similar situation occurs when protein is provided during endurance exercise, since blood flow to the muscle is also increased under these conditions thereby providing a rationale for mixed nutrient supplementation during endurance events to better support protein anabolism during recovery. Additive effects of exercise and BCAA supplementation on the activation of branched-chain ketoacid dehydrogenase were shown in a study by van Hall et al. (30), suggesting that BCAA oxidation may be accelerated even further by exogenous amino acid availability during exercise. Forslund (13) also demonstrated that high protein intake resulted in much greater rates of leucine oxidation when subjects exercised in both the fasted and fed state. Despite these observations, it is reasonable to propose that oxidation of the exogenous amino acids could spare free amino acids, which would then be available to the muscle during recovery. The fact that plasma concentrations of most essential amino acids were maintained or increased with MILK post exercise supports this notion. In summary, the present investigation of the metabolic response to low-energy carbohydrate and milk supplementation during prolonged exercise revealed a subtle influence of supplementary nutrients on the major energy regulating hormones. Supplements provided after the start of exercise, and therefore after initiation of the anticipatory endocrine response, must be supplied in fairly substantial quantities to reverse or suppress these events. Even in studies providing two to three times the amount of nutrients used in this protocol, alterations in the hormone response to endurance work are minimal and often do not persist throughout exercise (22, 28, 34). Energy provided to the subjects by the supplements was ~170 kcals divided into 4 portions, which were given at regular intervals throughout a 2-hour submaximal treadmill run. In response to this supplementation protocol, several aspects of the metabolic response to a 2-hour submaximal run differed due to carbohydrate (CHO) and mixed carbohydrate-protein (MILK) supplementation. CHO and MILK supplementation altered plasma concentrations of amino acids and free fatty acids, but not glucose. This observation demonstrates the high priority to maintain constant blood glucose levels under a variety of metabolic conditions. Supplementation influenced substrate utilization based on measures of RER, such that CHO and MILK inhibited the exercise-induced increase in FFA and promoted use of carbohydrate or non-lipid fuel during exercise. Since the MILK supplement was well-tolerated by the athletes in this study, it is possible that intact proteins might be incorporated into a carbohydrate-based sports drink designed to support protein utilization during recovery from training or competition. The intent of this study was to demonstrate how nutrient composition of a supplement consumed during exercise could influence the metabolic environment. MILK consumption during the run increased plasma amino acids following exercise and elicited a hormonal pattern associated with sparing endogenous carbohydrate and protein. While enhanced performance, as an acute response to supplementation or as a long-term consequence of improved recovery and body composition, was not examined in this investigation, study findings provide a rationale for continued investigation of the specific benefits of protein-based supplements for the endurance athlete.

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