Beetroot juice and exercise: pharmacodynamic and dose-response relationships

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1 J Appl Physiol 115: , First published My 2, 2013; doi: /jpplphysiol Beetroot juice nd exercise: phrmcodynmic nd dose-response reltionships Lee J. Wylie, 1 Jmes Kelly, 1 Stephen J. Biley, 1 Jmie R. Blckwell, 1 Philip F. Skib, 1 Pul G. Winyrd, 2 Asker E. Jeukendrup, 3 Anni Vnhtlo, 1 nd Andrew M. Jones 1 1 Sport nd Helth Sciences, College of Life nd Environmentl Sciences, University of Exeter, St. Luke s Cmpus, Exeter, United Kingdom; 2 University of Exeter Medicl School, St. Luke s Cmpus, Exeter, United Kingdom; nd 3 Gtorde Sports Science Institute, Brrington, Illinois Submitted 25 Mrch 2013; ccepted in finl form 25 April 2013 Wylie LJ, Kelly J, Biley SJ, Blckwell JR, Skib PF, Winyrd PG, Jeukendrup AE, Vnhtlo A, Jones AM. Beetroot juice nd exercise: phrmcodynmic nd dose-response reltionships. JApplPhysiol115: , First published My 2, 2013; doi: /jpplphysiol Dietry supplementtion with beetroot juice (BR), contining pproximtely 5 8 mmol inorgnic nitrte (NO 3 ), increses plsm nitrite concentrtion ([NO 2 ]), reduces blood pressure, nd my positively influence the physiologicl responses to exercise. However, the dose-response reltionship between the volume of BR ingested nd the physiologicl effects invoked hs not been investigted. In blnced crossover design, 10 helthy men ingested 70, 140, or 280 ml concentrted BR (contining 4.2, 8.4, nd 16.8 mmol NO 3, respectively) or no supplement to estblish the effects of BR on resting plsm [NO 3 ] nd [NO 2 ] over 24 h. Subsequently, on six seprte occsions, 10 subjects completed moderte-intensity nd severe-intensity cycle exercise tests, 2.5 h postingestion of 70, 140, nd 280 ml BR or NO 3 -depleted BR s plcebo (PL). Following cute BR ingestion, plsm [NO 2 ] incresed in dose-dependent mnner, with the pek chnges occurring t pproximtely 2 3 h. Compred with PL, 70 ml BR did not lter the physiologicl responses to exercise. However, 140 nd 280 ml BR reduced the stedy-stte oxygen (O 2 ) uptke during moderteintensity exercise by 1.7% (P 0.06) nd 3.0% (P 0.05), wheres time-to-tsk filure ws extended by 14% nd 12% (both P 0.05), respectively, compred with PL. The results indicte tht wheres plsm [NO 2 ] nd the O 2 cost of moderte-intensity exercise re ltered dose dependently with NO 3 -rich BR, there is no dditionl improvement in exercise tolernce fter ingesting BR contining 16.8 compred with 8.4 mmol NO 3. These findings hve importnt implictions for the use of BR to enhnce crdiovsculr helth nd exercise performnce in young dults. nitrte; nitrite; nitric oxide; blood pressure; exercise economy; O 2 uptke; exercise tolernce NITRIC OXIDE (NO) IS A GASEOUS signling molecule tht modultes humn physiologicl function vi its role in, for exmple, the regultion of blood flow, neurotrnsmission, immune function, glucose nd clcium homeostsis, muscle contrctility, nd mitochondril respirtion (9, 36). 1 NO is generted through the oxidtion of the mino cid L-rginine in rection ctlyzed by NO synthse (NOS), with nitrite (NO 2 ) nd nitrte (NO 3 ) being oxidtion products of NO (30). It is now pprecited tht under pproprite physiologicl conditions, NO cn lso be produced vi the reduction of NO 2, process tht my be prticulrly importnt in situtions where oxygen 1 This rticle is the topic of n Invited Editoril by L. Burke (5). Address for reprint requests nd other correspondence: A. M. Jones, College of Life nd Environmentl Sciences, Univ. of Exeter, St. Luke s Cmpus, Exeter EX1 2LU, UK (e-mil:.m.jones@exeter.c.uk). (O 2 ) vilbility is low, nd/or NOS function is impired (12). Interestingly, dministrtion of dietry inorgnic NO 3 hs been shown to increse plsm NO 2 concentrtion ([NO 2 ]) nd to produce NO-like bioctivity (19, 23, 39). Up to 25% of ingested NO 3 enters the enteroslivry circultion nd is concentrted in the sliv, whereupon fculttive, nerobic bcteri in the orl cvity reduce the NO 3 to NO 2 (30). When swllowed into the cidic environment of the stomch, some of the NO 2 is converted further into NO, wheres the reminder is bsorbed to increse circulting plsm [NO 2 ]. This NO 2 my be reduced further to NO nd other rective nitrogen intermedites, prticulrly in tissues tht my be reltively hypoxic, such s contrcting skeletl muscle (30). We (3, 37) nd others (19, 23, 39) hve demonstrted tht NO 3 ingestion, either in the form of NO 3 slts or vi the consumption of high NO 3 vegetble products, such s beetroot juice (BR), reduces resting blood pressure (BP) profoundly nd consistently. Consequently, dietry NO 3 supplementtion hs emerged s potentil nutritionl gent for the prevention nd tretment of hypertension nd crdiovsculr disese (30). Webb et l. (39) ssessed the effects of cute BR consumption (23 mmol NO 3 ) on plsm [NO 2 ] nd BP over 24 h. Plsm [NO 2 ] peked 3 h postingestion, remined close to pek vlues until 5 h postingestion, nd returned to bseline fter 24 h (39). The systolic nd distolic BP nd the men rteril pressure (MAP) were reduced significntly, by 10, 8, nd 8 mmhg, respectively, t h fter BR intke. The sme reserch group lter reported dose-dependent increse in plsm [NO 3 ] nd [NO 2 ] nd reduction in BP following ingestion of potssium NO 3 (KNO 3 ) (19). In this study, plsm [NO 2 ] rose by 1.3-, pproximtely two-, nd pproximtely fourfold following consumption of 4, 12, nd 24 mmol KNO 3, respectively. The pek rise in plsm [NO 2 ] ws ccompnied by significnt reductions in both systolic BP (of 2, 6, nd 9 mmhg, respectively) nd distolic BP (of 4, 4, nd 6 mmhg, respectively). However, since BR contins polyphenols nd ntioxidnts, which cn fcilitte the synthesis of NO from NO 2 in the stomch (30), it is uncler whether BP is similrly impcted when different doses of BR re ingested compred with equivlent doses of NO 3 slts. Given the growing interest in dietry NO 3 supplementtion in the form of BR mongst thletes nd the generl popultion, it is importnt to determine the phrmcokinetic-phrmcodynmic reltionship between different volumes of BR consumption nd chnges in plsm [NO 2 ] nd BP to estblish n optiml dose for beneficil effects. Recent investigtions suggest tht dietry NO 3 supplementtion hs the potentil to influence humn physiology beyond /13 Copyright 2013 the Americn Physiologicl Society 325

2 326 Nitrte nd Exercise Dose Response Wylie LJ et l. the bove hemodynmic effects (3, 26). Specificlly, we (2, 3, 22) nd others (6, 24 26) hve demonstrted tht 3 6 dys of dietry NO 3 supplementtion reduces the O 2 cost of moderteintensity exercise nd my enhnce exercise tolernce in helthy, young dults. It ppers tht these effects re relted to NO 2 or NO-medited enhncements of muscle contrctile function (2, 17) nd/or mitochondril efficiency (24) nd/or enhnced muscle blood flow, especilly to type II fibers (14). Importntly, reduction of the O 2 cost of exercise (25, 37) nd improved exercise performnce (21) hs lso been reported s erly s 2.5 h following single dose of dietry NO 3, which is consistent with the time required for the pek plsm [NO 2 ] to be ttined (39). However, since ll exercise-performnce studies completed to dte with BR hve dministered pproximtely 5 8 mmol NO 3, it is uncler whether dose-response reltionship exists between cute NO 3 intke nd the physiologicl responses to exercise. The estblishment of the doseresponse reltionship between NO 3 intke nd the physiologicl responses to exercise nd the scertinment of the optiml NO 3 dose for enhncing exercise performnce re importnt, given the incresing populrity of BR supplementtion in both bsic reserch nd pplied exercise settings. Therefore, the purpose of the present study ws twofold: firstly, to chrcterize the plsm [NO 3 ] nd [NO 2 ] phrmcokinetics nd the chnges in BP fter ingestion of three different quntities of NO 3 -rich BR; nd secondly, to investigte the dose-response reltionship between BR/NO 3 intke nd the physiologicl responses to exercise. In two seprte experiments, we dministered BR concentrte tht enbled substntil NO 3 lod to be ingested quickly nd esily. We investigted: 1) the influence of cute NO 3 doses of 4.2, 8.4, nd 16.8 mmol consumed in 70, 140, nd 280 ml concentrted BR on plsm [NO 3 ] nd [NO 2 ] nd BP over 24-h period; nd 2) the physiologicl responses to step trnsitions to moderte- nd severe-intensity exercise, 2.5 h postingestion of the sme NO 3 doses. We hypothesized tht the effects of dietry inorgnic NO 3 on plsm [NO 3 ] nd [NO 2 ], BP, the O 2 cost of moderte-intensity exercise, nd exercise tolernce (ssessed s the time-to-tsk filure) during severe-intensity exercise would be dose dependent. METHODS The study ws conducted in two phses [study 1 (S 1), phrmcokinetics; nd S 2, dose response], with the results generted in S 1 used to inform the experimentl design in S 2. There ws distinct subject recruitment for ech experiment. Ten helthy, recretionlly ctive men volunteered for ech experiment [men SD: S 1, ge 23 5 yr, height m, body mss (BM) 79 9 kg; S 2, ge 22 5 yr, height m, BM 74 8 kg]. None of the subjects in S 1 nd S 2 ws tobcco smoker or user of dietry supplements. All subjects recruited for S 2 were fully fmilir with lbortory exercise-testing procedures, hving prticipted previously in studies using cycle ergometry in our lbortory. The procedures used in S 1 nd S 2 were grnted full ethics pprovl by the Institutionl Reserch Ethics Committee. All subjects gve their written, informed consent to prticipte fter the experimentl procedures, ssocited risks, nd potentil benefits of prticiption hd been explined in detil. All subjects in S 1 nd S 2 were instructed to keep food nd physicl-ctivity diry in the 24 h preceding their first lbortory visit nd to replicte food consumption nd physicl ctivity in the 24 h preceding subsequent visits. The subjects were required to rrive t the lbortory in rested nd fully hydrted stte, following n overnight fst, nd to void strenuous ctivity in the 24 h preceding ech testing session. Subjects were instructed to refrin from cffeine nd lcohol-contining drinks for 6 nd 24 h before ech lbortory visit, respectively, nd to bstin from using ntibcteril mouthwsh nd chewing gum throughout the study, becuse these re known to erdicte the orl bcteri tht re necessry for the conversion of NO 3 to NO 2 (16). S 1: Phrmcokinetics nd Phrmcodynmics Procedures. All subjects reported to the lbortory on four seprte occsions over period of 3 wk. Upon rrivl to the lbortory, resting BP ws mesured, nd venous blood smple ws obtined for the mesurement of plsm [NO 2 ] nd [NO 3 ]. Subjects then consumed n cute dose of 70, 140, or 280 ml NO 3 -rich BR (orgnic BR contining 4.2, 8.4, or 16.8 mmol NO 3, respectively; Beet It; Jmes White Drinks, Ipswich, UK) or 140 ml wter [control (CON)], in ddition to stndrdized brekfst (72 g porridge ots with 180 ml semiskimmed milk). BP ws mesured, nd venous blood smple ws obtined, 1, 2, 4, 8, 12, nd 24 h postingestion. For ech 24-h period of dt collection, subjects were provided with stndrdized, low NO 3 diet. The quntity nd timing of food nd drink intke were recorded on visit 1 nd replicted in subsequent visits. A wshout period of t lest 3 dys seprted the lbortory visits. Mesurements. The BP of the brchil rtery ws mesured using n utomted sphygmomnometer (Dinmp Pro; GE Medicl Systems, Tmp, FL), with the subjects in seted position. After rrivl t the lbortory nd following 10 min of rest in n isolted room, four mesurements were recorded, nd the men of the finl three mesurements ws used for dt nlysis. Venous blood smples were drwn into lithium-heprin tubes (7.5 ml Monovette lithium heprin; Srstedt, Leicester, UK). Smples were centrifuged t 4,000 rpm nd 4 C for 7 min, within 1 min of collection. Plsm ws subsequently extrcted nd immeditely frozen t 80 C for lter nlysis of [NO 2 ] nd [NO 3 ]. All glsswre, utensils, nd surfces were rinsed with deionized wter to remove residul [NO 2 ] nd [NO 3 ] before blood nlyses. The [NO 2 ] of the undiluted (nondeproteinized) plsm ws determined by its reduction to NO in the presence of glcil cetic cid nd 4% (w/v) queous sodium iodide. The spectrl emission of electroniclly excited nitrogen dioxide product, from the NO rection with ozone, ws detected by thermoelectriclly cooled, red-sensitive photomultiplier tube, housed in Sievers gs-phse chemiluminescence NO nlyzer (NOA; Sievers NOA 280i; Anlytix, Durhm, UK). The [NO 2 ] ws determined by plotting signl (mv) re ginst clibrtion plot of 100 nm 1 M sodium NO 2. Before determintion of [NO 3 ], smples were deproteinized using zinc sulfte (ZnSO 4)/sodium hydroxide (NOH) precipittion. Aqueous ZnSO 4 [400 l 10% (w/v)] nd 400 l 0.5 M NOH were dded to 200 l of smple nd vortexed for 30 s before being left to stnd t room temperture for 15 min. Therefter, smples were centrifuged t 4,000 rpm for 5 min, nd the superntnt ws removed for subsequent nlysis. The [NO 3 ] of the deproteinized plsm smple ws determined by its reduction to NO in the presence of 0.8% (w/v) vndium trichloride in 1 M HCl. The production of NO ws detected using the chemiluminescence NOA, s described bove. To determine more precisely the time-to-pek plsm [NO 2 ] following NO 3 ingestion, one-comprtment model with first-order bsorption nd elimintion kinetics ws used, s described in the following eqution Y exp(ke X (Ke K) expke X) (Ke K 1) where Y represents frction bsorbed; X represents time; nd, K nd Ke represent the first-order bsorption nd elimintion rte constnts, respectively. Sttisticl nlysis. Two-wy repeted-mesures ANOVA ws used to ssess the difference cross conditions (4.2, 8.4, nd 16.8 J Appl Physiol doi: /jpplphysiol

3 Nitrte nd Exercise Dose Response Wylie LJ et l. 327 mmol NO 3 nd CON) nd cross time (0, 1, 2, 4, 8, 12, nd 24 h) for plsm [NO 2 ] nd [NO 3 ] nd BP. Significnt min or interction effects were nlyzed further using simple contrsts. One-wy repetedmesures ANOVA ws used to ssess the differences in time-to-pek plsm [NO 2 ]. Reltionships between plsm [NO 2 ] nd BP were nlyzed using Person product moment correltion coefficients. Sttisticl significnce ws ccepted t P Results re presented s men SD unless stted otherwise. S 2: Dose Response Protocol. Subjects were required to report to the lbortory on seven seprte occsions, over 4- to 5-wk period. During the first visit to the lbortory, subjects completed rmp incrementl exercise test for determintion of pek O 2 uptke (V O 2 pek) nd gs-exchnge threshold (GET). All tests were performed on n electroniclly brked cycle ergometer (Lode Exclibur Sport, Groningen, The Netherlnds). Initilly, ech subject completed 3 min of unloded bseline cycling; then, the work rte ws incresed by 30 W/min until the subject ws unble to continue. The subjects cycled t self-selected pedl rte (70 90 rpm), nd this pedl rte long with the sddle nd hndlebr height nd configurtion were recorded nd reproduced in subsequent tests. The breth-by-breth pulmonry gs-exchnge dt were collected continuously during the incrementl tests nd verged over consecutive 10-s periods. V O 2 pek ws tken s the highest 30-s men vlue ttined before the subject s volitionl exhustion. The GET ws determined s described previously (3, 37). The work rtes tht would require 80% of the GET (moderte-intensity exercise) nd 75% of the difference between the power output t GET nd V O 2 pek plus the power output t GET, i.e., severe-intensity exercise () were subsequently clculted. On test dys, subjects rrived t the lbortory t 8 AM. A venous blood smple ws drwn for mesurement of plsm [NO 2 ] nd NO 3. Subjects then ingested 70, 140, or 280 ml NO 3 -rich BR (contining 4.2, 8.4, or 16.8 mmol NO 3, respectively; Beet It) or 70, 140, or 280 ml NO 3 -depleted BR s plcebo (PL70, PL140, or PL280; contining 0.04, 0.08, or 0.12 mmol NO 3 ; Beet It). All BR nd PL doses were dministered using rndomized, double-blind crossover design. Subjects were sked to consume the beverge within 5-min period nd, fter doing so, were served stndrdized brekfst (72 g porridge with 180 ml semiskimmed milk). A wshout period of t lest 72 h seprted ech visit. After ingestion of the beverge, subjects were given period of 2.5 h, during which they were llowed to leve the lbortory but were sked to refrin from strenuous physicl ctivity. Subjects were lso sked to fst during this time, lthough wter ws permitted d libitum. Following this 2.5-h period, second venous blood smple ws drwn for mesurement of plsm [NO 2 ] nd [NO 3 ]. Subjects then completed step exercise tests, from 20-W bseline to moderte-intensity (93 11 W) nd severe-intensity ( W) work rtes for the determintion of pulmonry V O 2 dynmics. On ech visit, subjects completed two, 5-min bouts of moderte-intensity exercise nd one bout of severe-intensity exercise tht ws continued until tsk filure s mesure of exercise tolernce. All bouts of exercise on ech dy were seprted by 5 min of pssive rest. The time-to-tsk filure ws recorded when the pedl rte fell by 10 rpm below the self-selected pedl rte. In the severe-intensity bouts, the subjects were verblly encourged to continue for s long s possible. Mesurements. During ll exercise tests, pulmonry gs exchnge nd ventiltion were mesured breth by breth, with subjects wering nose clip nd brething through low ded-spce (90 ml), lowresistnce (0.75 mmhg1 1 s 1 t 15 l/s) mouthpiece nd impeller turbine ssembly (Jeger Triple-V; Jeger GmbH, Hoechberg, Germny). The inspired nd expired gs volume nd gs concentrtion signls were smpled continuously t 100 Hz the ltter using prmgnetic (O 2) nd infrred [crbon dioxide (CO 2)] nlyzers (Oxycon Pro; Jeger GmbH) vi cpillry line connected to the mouthpiece. These nlyzers were clibrted before ech test with gses of known concentrtion, nd the turbine volume trnsducer ws clibrted using 3-liter syringe (Hns Rudolph, Knss City, MO). The volume nd concentrtion signls were time ligned by ccounting for the dely in cpillry gs trnsit nd nlyzer rise time reltive to volume signl. O 2 uptke, CO 2 output, nd minute ventiltion were clculted using stndrd formul nd displyed breth by breth. Hert rte (HR) ws mesured using short-rnge rdiotelemetry (model RS400; Polr Electro Oy, Kempele, Finlnd). Cpillry blood smples were collected from the fingertip into cpillry tube during the bseline, preceding ech step trnsition in work rte; during the finl 30 s of ech moderte-intensity exercise bout; nd following exhustion in the severe-intensity exercise bout. These smples were nlyzed immeditely to determine blood lctte concentrtion ([lctte]; model YSI 1500; Yellow Springs Instrument, Yellow Springs, OH). Venous blood smples were treted nd nlyzed s described in S 1. The breth-by-breth dt from ech exercise test were linerly interpolted to provide second-by-second vlues, nd the two identicl, moderte-intensity repetitions performed on ech visit were time ligned to the strt of exercise nd ensemble verged. Bseline V O 2 (V O 2bseline), expired CO 2 t bseline (V CO 2bseline), nd respirtory exchnge rtio (RER) t bseline were defined s the men vlues mesured over the finl 90 s of bseline pedling. The end-exercise V O 2,V CO 2, nd RER were defined s the men vlues mesured over the finl 30 s of exercise. The mplitude of the V O 2 response ws clculted by subtrcting V O 2bseline from V O 2 t the end of exercise. Subsequently, the functionl gin of the entire response ws clculted by dividing the V O 2 mplitude by the chnge () in work rte. The mplitude of the V O 2 slow component during the severe-intensity exercise bout ws estimted by subtrcting the men V O 2 t 2 min from the men V O 2 t 6 min. Sttisticl nlysis. Two-wy repeted-mesures ANOVA ws used to ssess the difference in pulmonry gs-exchnge vribles, blood [lctte], nd HR cross dose (70, 140, nd 280 ml) nd tretment (PL nd BR). Differences in pre- nd postplsm [NO 2 ] nd [NO 3 ] were ssessed seprtely in PL nd BR, cross dose nd time (pre nd post) using two-wy repeted-mesures ANOVAs. Significnt min nd interction effects were nlyzed further using simple contrsts. Sttisticl significnce ws ccepted t P Results re presented s men SD unless stted otherwise. RESULTS Ingestion of BR ws tolerted well by ll subjects in S 1 nd S 2. Subjects did, however, report beeturi (red urine) nd red stools, consistent with previous studies (3, 39). The bsolute NO 3 doses used in S 1 nd S 2 (4.2, 8.4, nd 16.8 mmol) were equivlent to (rnge: ), (rnge: ), nd mmol (rnge: ) NO 3 /kg BM, respectively. S 1 : Phrmcokinetics nd Phrmcodynmics The effects of different volumes of BR (nd therefore, different mounts of ingested NO 3 ) on plsm [NO 3 ] nd [NO 2 ] re presented in Fig. 1. There were significnt min effects by dose nd time nd n interction effect for both plsm [NO 3 ] (Fig. 1A; ll P 0.01) nd plsm [NO 2 ] (Fig. 1B; ll P 0.01). At resting bseline, before the ingestion of ny beverge, plsm [NO 3 ] ws not significntly different between doses (Fig. 1A; ll P 0.05). ANOVA nlyses reveled significnt dose-dependent increses in plsm [NO 3 ] following BR supplementtion (P 0.05). The pek elevtion bove bseline in plsm [NO 3 ] occurred 1 h postdministrtion of 4.2 (160 J Appl Physiol doi: /jpplphysiol

4 328 Nitrte nd Exercise Dose Response Wylie LJ et l. A b c b c b c 500 Fig. 1. Plsm nitrte concentrtion ([NO 3 ]; A) nd nitrite concentrtion ([NO 2 ]; B) following consumption of wter (control; ) nd 4.2 (Œ), 8.4 (), nd 16.8 (}) mmol NO 3 (group men SE). Plsm [NO 3 ] nd [NO 2 ] rose significntly in dose-dependent mnner. See text for further detils. Significnt difference from presupplemention bseline (P 0.05); significnt difference from control (P 0.05); b significnt difference from 4.2 mmol NO 3 (P 0.05); c significnt difference from 8.4 mmol NO 3 (P 0.05). Plsm [NO 2 - ](nm) Plsm [NO 3 - ](μm) B b b b b c b b c b b b c b b c b b c b b b b c b b c Time (h) 43 M) nd 8.4 mmol NO 3 ( M) nd 2 h postdministrtion of 16.8 mmol NO 3 ( M; Fig. 1A; ll P 0.05). Plsm [NO 3 ] remined elevted bove bseline nd CON t ll time points fter dministrtion of 4.2, 8.4, nd 16.8 mmol NO 3 (P 0.05). At bseline, before ingestion of ny beverge, plsm [NO 2 ] ws not significntly different between doses (Fig. 1B; P 0.05). ANOVA nlyses reveled significnt dose-dependent increses in plsm [NO 2 ] following BR supplementtion (P 0.05). The pek elevtion bove bseline in plsm [NO 2 ] occurred 2 h postdministrtion of 4.2 ( nm) nd 8.4 mmol NO 3 ( nm) nd 4 h postdministrtion of 16.8 mmol NO 3 ( nm; Fig. 1B; ll P 0.05). Kinetic nlyses reveled tht plsm [NO 2 ] peked significntly lter ( min; rnge: min) following ingestion of 16.8 mmol reltive to both 8.4 mmol ( min; rnge: min; P 0.05) nd 4.2 mmol BR ( min; rnge: min; P 0.05). Pek plsm [NO 2 ], following ingestion of 8.4 mmol, tended to occur lter compred with 4.2 mmol (P 0.06). Plsm [NO 2 ] remined elevted bove bseline nd CON t 1, 2, 4, nd 8 h fter dministrtion of 4.2, 8.4, nd 16.8 mmol NO 3 (ll P 0.05). At 12 h, plsm [NO 2 ] remined elevted bove bseline nd 4.2 mmol BR following ingestion of 8.4 nd 16.8 mmol NO 3 (ll P 0.05). In ddition, plsm [NO 2 ] remined elevted t 24 h following dministrtion of 16.8 mmol NO 3 compred with ll other doses (P 0.05). The effects of different volumes of BR (nd therefore, different mounts of ingested NO 3 ) on systolic nd distolic BP nd MAP re presented in Fig. 2. The chnges in systolic BP cross ll conditions re presented in Fig. 2A. There were significnt min effects by dose nd time nd n interction effect on systolic BP (ll P 0.05). Systolic BP t bseline, before dministrtion of ny beverge, ws lower (P 0.05) in the 16.8-mmol NO 3 condition (118 5mmHg)reltivetoCON (121 5mmHg)butnotreltiveto4.2(119 6 mmhg) nd 8.4 mmol NO 3 (120 6 mmhg). Compred with bseline, systolic BP ws lowered significntly following ingestion of 4.2, 8.4, nd 16.8 mmol NO 3 (ll P 0.05). The pek reduction in systolic BP occurred 4 h postdministrtion of 4.2 (5 5 mmhg), 8.4 (10 5 mmhg), nd 16.8 mmol NO 3 (9 4 mmhg), respectively, reltive to bseline (ll P 0.05). Systolic BP ws reduced reltive to bseline, CON, nd 4.2 mmol NO 3, t 2, 4, nd 8 h postdministrtion of 8.4 mmol nd 16.8 mmol NO 3 (ll P 0.05). There were no differences in systolic BP between 8.4 nd 16.8 mmol NO 3 t ny time point (P 0.05). At 24 h, systolic BP remined significntly lower (by 5 5 mmhg) thn bseline, following consumption of 16.8 mmol NO 3 (P 0.05). In contrst, systolic BP ws not significntly different thn CON or bseline t 24 h postd- J Appl Physiol doi: /jpplphysiol

5 Nitrte nd Exercise Dose Response Wylie LJ et l. 329 A Δ MAP (mmhg) Δ Distolic BP (mmhg) Δ Systolic BP (mmhg) B C b b b b b b Time (h) Fig. 2. Chnge () reltive to presupplementtion bseline in systolic blood pressure (BP; A), distolic BP (B), nd men rteril pressure (MAP; C) following consumption of wter (control; ) nd 4.2 (Œ), 8.4 (), nd 16.8 (}) mmol NO 3 (group men SE). Significnt difference from presupplemention bseline (P 0.05); significnt difference from control (P 0.05); b significnt difference from 4.2 mmol NO 3 (P 0.05). ministrtion of 4.2 nd 8.4 mmol NO 3 (P 0.05). Overll, the men systolic BP cross 24 h, reltive to CON, ws lowered dose dependently by 3, 4, nd 6 mmhg fter dministrtion of 4.2, 8.4, nd 16.8 mmol NO 3, respectively (ll P 0.05). The chnge in systolic BP ws correlted with the chnge in plsm [NO 3 ](r 0.27; P 0.05) nd the chnge in plsm [NO 2 ](r 0.37; P 0.05). The pek reduction in systolic BP ws not correlted with the bseline systolic BP. The chnges in distolic BP following the ingestion of different doses of NO 3 -rich BR re presented in Fig. 2B. There ws significnt interction effect (dose time) on distolic BP (P 0.05). Distolic BP t bseline ws not significntly different mong conditions (CON: 67 5; 4.2 mmol: 68 4; 8.4 mmol: 68 6; 16.8 mmol: 67 6 mmhg; P 0.05). Follow-up tests reveled tht ingestion of 8.4 nd 16.8 but not 4.2 mmol NO 3 reduced distolic BP significntly, reltive to bseline nd CON (ll P 0.05). The pek reduction in distolic BP from bseline occurred t 4 h postdministrtion of 8.4 mmol NO 3 (3 3 mmhg) nd 2 h postdministrtion of 16.8 mmol NO 3 (4 4 mmhg; both P 0.05) reltive to bseline (both P 0.05) nd returned to ner-bseline vlues by 24 h (P 0.05). There were no differences in distolic BP between 8.4 nd 16.8 mmol NO 3 t ny time point (P 0.05). The chnge in distolic BP ws correlted with the chnge in plsm [NO 3 ](r 0.35; P 0.05) nd the chnge in plsm [NO 2 ](r 0.39; P 0.05). Moreover, the pek chnge in distolic BP ws correlted with the bseline distolic BP (r 0.49; P 0.05). The chnges in MAP following the ingestion of different doses of NO 3 -rich BR re presented in Fig. 2C. There were significnt min effects by dose nd time nd n interction effect on MAP (ll P 0.05). At bseline, before the ingestion of ny beverge, MAP ws not significntly different mong conditions (CON: 85 4; 4.2 mmol: 85 4; 8.4 mmol: 85 5; 16.8 mmol: 84 5 mmhg; P 0.05). MAP ws significntly lower following ingestion of 4.2, 8.4, nd 16.8 mmol NO 3 reltive to bseline nd CON (ll P 0.05). Following ingestion of 4.2 mmol NO 3, the pek reduction (2 2 mmhg) in MAP occurred t 1 h, nd MAP remined reduced by 2 mmhg t 2 h reltive to bseline (P 0.05). In contrst, the pek reduction in MAP (5 3 mmhg) occurred 4 h postdministrtion of 8.4 nd 16.8 mmol NO 3 reltive to bseline (P 0.05). MAP ws not different between 8.4 nd 16.8 mmol NO 3 t ny time point (P 0.05). Overll, the men MAP cross 24 h, reltive to CON, ws reduced dose dependently by 1, 2, nd 4 mmhg fter dministrtion of 4.2, 8.4, nd 16.8 mmol NO 3, respectively (ll P 0.05). The chnge in MAP ws correlted significntly with the chnge in plsm [NO 3 ](r 0.35; P 0.05) nd the chnge in plsm [NO 2 ] (r 0.41; P 0.05). S 2 : Dose Response Plsm [NO 3 ] nd [NO 2 ]. The group men plsm [NO 3 ] nd [NO 2 ] responses in the BR nd PL conditions re illustrted in Fig. 3, A nd B, respectively. Presupplementtion plsm [NO 3 ] ws not significntly different between conditions (P 0.05), nd no significnt chnge in plsm [NO 3 ] ws observed following PL supplementtion (P 0.05). ANOVA nlyses reveled significnt dose-dependent increse in plsm [NO 3 ] t 2.5 h following BR supplementtion (P 0.05). An elevtion in plsm [NO 3 ] bove bseline ws pprent following 4.2 ( M; P 0.05), 8.4 ( M; P 0.05), nd 16.8 mmol NO 3 ( M; P 0.05). Presupplementtion plsm [NO 2 ] ws not significntly different mong conditions (P 0.05), nd no significnt chnge in plsm [NO 2 ] ws observed following PL supplementtion (P 0.05). ANOVA nlyses reveled significnt dose-dependent increse in plsm [NO 2 ] t 2.5 h following BR supplementtion (P 0.05). Following dministrtion of 4.2, 8.4, nd 16.8 mmol NO 3, plsm [NO 2 ] ws elevted bove bseline by nm, nm, nd nm, respectively (ll P 0.05). Plsm [NO 2 ] ws significntly greter fter ingestion of 16.8 mmol compred with 4.2 mmol NO 3 (P 0.05) nd tended to be greter compred with 8.4 mmol NO 3 (P 0.06). Plsm [NO 2 ] ws J Appl Physiol doi: /jpplphysiol

6 330 Nitrte nd Exercise Dose Response Wylie LJ et l. A Plsm [NO 3 - ](μm) B Plsm [NO2 - ](nm) b 70 ml 140 ml 280 ml 70 ml 140 ml 280 ml Nitrte Plcebo 70 ml 140 ml 280 ml 70 ml 140 ml 280 ml Nitrte Plcebo Fig. 3. Men SE plsm [NO 3 ](A) nd [NO 2 ](B) preingestion (blck brs) nd 2.5-h postingestion (gry brs) of 70, 140, nd 280 ml NO 3 -rich beetroot juice (BR) (NO 3 ) or NO 3 -depleted BR [plcebo (PL)]. See text for further detils. Significnt difference from bseline (P 0.05); significnt difference postconsumption of 70 ml NO 3 -rich BR (P 0.05); b significnt difference from postconsumption of 140 ml NO 3 -rich BR (P 0.05). significntly greter following ingestion of 8.4 mmol NO 3 compred with 4.2 mmol NO 3 (P 0.05). Moderte-intensity exercise. The pulmonry gs exchnge nd ventiltory responses to moderte-intensity exercise cross ll doses nd conditions re summrized in Tble 1. The V O 2 mesured during the period of bseline cycling t 20 W ws not ffected by dose or condition (P 0.05). However, the bsolute end-exercise V O 2, mesured over the finl 30 s of moderte-intensity exercise, ws ltered significntly by BR ingestion (P 0.05; Fig. 4A). Follow-up tests indicted tht end-exercise V O 2 ws lowered significntly by 3% following dministrtion of 16.8 mmol NO 3 reltive to the respective PL (PL280: vs. BR280, l/min; P 0.05). In ddition, there ws trend towrd significnt reduction (2%) in end-exercise V O 2 following dministrtion of 8.4 mmol NO 3 reltive to the respective PL (PL140: vs. BR140, l/min; P 0.06). The chnge in plsm [NO 2 ] from bseline to postingestion of 4.2, 8.4, nd 16.8 mmol NO 3 ws correlted with the chnge in endexercise V O 2 (r 0.47; P 0.05). There ws no significnt difference in end-exercise V O 2 following ingestion of 4.6 mmol NO 3 (BR70) compred with PL70 (P 0.05). The mplitude of the V O 2 response (end-exercise V O 2bseline ; Tble 1) ws ffected by dose (P 0.05) nd tended to be ffected by condition (P 0.07). Follow-up tests reveled tht there ws trend towrd significnt reduction in the V O 2 mplitude (by 6%) fter dministrtion of 16.8 mmol NO 3 compred with 8.4 mmol NO 3 (BR140: vs. BR280: l/min; P 0.06). The chnge in plsm [NO 2 ] from bseline to postingestion of 4.2, 8.4, nd 16.8 mmol NO 3 ws correlted with the chnge in V O 2 mplitude (r 0.38; P 0.05). There ws no significnt difference in V O 2 mplitude between PL nd BR t ny dose (P 0.05). The V CO 2bseline, mesured over the lst 90 s of 20 W pedling, nd the end-exercise V CO 2, mesured over the lst 30 s of exercise, were ffected by dose (P 0.05 for both) but not condition (P 0.05 for both; Tble 1). Follow-up tests reveled tht V CO 2bseline ws incresed significntly, s the volume of supplement ingested incresed (P 0.05), irrespective of the condition (i.e., PL or BR). Specificlly, V CO 2bseline ws incresed by 7% nd 5% following consumption of 280 ml of supplement reltive to 70 nd 140 ml, respectively (P 0.05 for both). There were no significnt differences in V CO 2 between the ingestion of 70 nd 140 ml of supplement (P 0.05). Furthermore, post hoc nlysis reveled tht the end-exercise V CO 2 ws significntly higher following ingestion of both 140 nd 280 ml of supplement reltive to 70 ml (P 0.01 for both). There ws, however, no significnt difference in end-exercise V CO 2 between ingestion of 140 nd 280 ml of supplement (P 0.05). Bseline nd end-exercise RER were ffected by dose (P 0.05 for both) but not condition (P 0.05). The follow-up tests indicted tht RER incresed s the volume of supplement ingested incresed (P 0.05; Tble 1). Specificlly, RER t bseline ws incresed by 5% nd 4%, following consumption of 280 ml of supplement reltive to 70 nd 140 ml, respectively (P 0.05 for both). Although there ws no significnt interction effect or min effect by condition, bseline RER tended to be higher (by 3%) following dministrtion of 16.8 mmol NO 3 compred with the respective PL (P 0.08). End-exercise RER ws incresed significntly by 4% nd 3%, following consumption of 280 ml compred with 70 nd 140 ml of supplement, respectively (P 0.05 for both). In ddition, the ingestion of 140 ml incresed end-exercise RER compred with ingestion of 70 ml of supplement (P 0.05). The bseline, end-exercise, nd chnge in blood [lctte] nd HR were not ltered significntly by dose or condition (Tble 2; P 0.05). Severe-intensity exercise. The pulmonry gs exchnge nd ventiltory responses to severe-intensity exercise cross ll doses nd conditions re summrized in Tble 1. In contrst to the effects observed for moderte-intensity exercise, the V O 2 nd V CO 2 mesured t bseline nd t tsk filure were not ltered by dose or tretment (ll P 0.05). Moreover, neither the dose nor the tretment ltered the V O 2 slow component mplitude (P 0.05 for both). There ws trend towrd significnt min effects by dose (P 0.09) nd tretment (P 0.08) but no interction effect on RER t bseline (P 0.05). Follow-up tests reveled tht there ws trend towrd significnt increses in RER t bseline by 4% nd 3% following consumption of 280 ml of supplement compred with the consumption of 70 (P 0.06) or 140 ml (P 0.08) of supplement, respectively. RER, t tsk filure, ws not ltered by dose or tretment (P 0.05). The bseline, end-exercise, nd chnge in blood [lctte] nd HR were not ltered significntly by dose or condition (Tble 2; P 0.05). There ws significnt min effect by condition (P 0.05) but not dose (P 0.05) on time-to-tsk filure (Tble 1 nd J Appl Physiol doi: /jpplphysiol

7 Nitrte nd Exercise Dose Response Wylie LJ et l. Tble 1. Pulmonry gs-exchnge vribles during moderte- nd severe-intensity exercise following supplementtion with 3 different volumes of beetroot juice nd plcebo 70 ml 140 ml 280 ml Plcebo Nitrte, 4.2 mmol Plcebo Nitrte, 8.4 mmol Plcebo Nitrte, 16.8 mmol Moderte-intensity exercise V O 2 Bseline, l/min End-exercise, l/min b Primry mplitude, l/min Primry gin, ml min 1 W c V CO 2 Bseline, l/min ,d End-exercise, l/min d V E Bseline, l/min End-exercise, l/min RER Bseline c,d End-exercise c,d Severe-intensity exercise V O 2 Bseline, l/min End-exercise, l/min Overll gin, ml min 1 W Slow-phse mplitude, 6 2 min; l/min V CO 2 Bseline, l/min End-exercise, l/min RER Bseline End-exercise Time-to-tsk filure(s) e b Vlues re mens SD. V O 2, oxygen uptke; V CO 2, expired crbon dioxide; V E, ventiltion; RER, respirtory exchnge rtio. Significntly different from plcebo (PL)70 (P 0.05); b significntly different from PL280 (P 0.05); c Significntly different from beetroot juice (BR)140 (P 0.05); d significntly different from BR70 (P 0.05); e significntly different from PL140 (P 0.05). 331 Fig. 4B). Follow-up tests reveled tht consumption of 8.4 mmol NO 3 (BR140) nd 16.8 mmol NO 3 (BR280) resulted in significnt increse in time-to-tsk filure by s nd s, respectively, reltive to PL140 nd PL280 (P 0.05; Fig. 4B). There ws no difference in time-to-tsk filure between BR70 nd PL70 (P 0.05). The chnge in plsm [NO 2 ] from bseline to postingestion of 4.2, 8.4, nd 16.8 mmol NO 3 ws correlted significntly with the chnge in time-to-tsk filure (r 0.55; P 0.05). There ws no significnt difference in time-to-tsk filure mong 4.2, 8.4, nd 16.8 mmol BR (ll P 0.05) or mong PL70, PL140, nd PL280 (P 0.05). In terms of positive chnges in time-to-tsk filure, there were three nonresponders in the 4.2-mmol condition, two in the 8.4-mmol condition, nd one in the 16.8-mmol condition. Individul subjects who did not respond t lower doses did respond t higher doses. The increse in plsm [NO 2 ] from bseline to pre-exercise for the nonresponders ws similr to the other subjects who did respond. For exmple, the three nonresponders t the lowest NO 3 dose hd n increse in plsm [NO 2 ] of 140, 208, nd 161 nm compred with group men increse of 150 nm. In ddition, the nonresponders did not hve high bseline vlues of plsm [NO 2 ] ( nm) compred with the group men. DISCUSSION This study is the first to chrcterize the phrmcokineticphrmcodynmic effects of NO 3 -rich BR ingestion nd to investigte the dose-response reltionship between BR ingestion nd the physiologicl responses to exercise. Specificlly, we studied how cute ingestion of three different BR volumes (nd thus three different NO 3 doses) impcted on plsm [NO 3 ] nd [NO 2 ], resting BP, the pulmonry gs-exchnge responses to moderte- nd severe-intensity exercise, nd exercise tolernce. Our principl findings were tht plsm [NO 3 ] nd [NO 2 ] incresed dose dependently up to 16.8 mmol NO 3 with there being dose-dependent pek reduction in BP up to 8.4 mmol NO 3. A NO 3 dose of 16.8 mmol ws required to elicit significnt reduction in the O 2 cost of moderte-intensity cycle exercise, lthough there ws trend (P 0.06) for reduction with 8.4 mmol. A significnt improvement in time-to-tsk filure during severeintensity exercise ws evident fter ingestion of 8.4 mmol NO 3,withnofurtherbenefitsobservedfollowingtheingestion of 16.8 mmol NO 3. S 1 : BR Phrmcokinetics nd Phrmcodynmics Effects on Plsm [NO 3 ], [NO 2 ], nd BP The results of S 1 demonstrted tht concentrted BR consumption cuses dose-dependent increses in plsm [NO 3 ] nd [NO 2 ]. Plsm [NO 3 ] incresed by pproximtely fivend eightfold, 1 h fter the ingestion of 4.2 nd 8.4 mmol NO 3, nd by 18-fold, 2 h fter the ingestion of 16.8 mmol NO 3. In contrst, the increse in plsm [NO 2 ] occurred lter, peking t pproximtely h postdministrtion of 4.2 nd 8.4 mmol NO 3 nd 3 h postdministrtion of 16.8 mmol NO 3. J Appl Physiol doi: /jpplphysiol

8 332 Nitrte nd Exercise Dose Response Wylie LJ et l. A End-exercise o 2 (L.min -1 ). B Time to Tsk Filure (s) PL BR 70 ml PL BR 140 ml As expected, the rise in plsm [NO 2 ] ws smller compred with plsm [NO 3 ], with pek increses of 2.5-fold, pproximtely fourfold, nd pproximtely eightfold, respectively. The delyed pek increses in plsm [NO 2 ] compred with plsm [NO 3 ] reflect the importnce of the enteroslivry circultion nd subsequent reduction of NO 3 to NO 2 by PL BR 280 ml Fig. 4. Men SE stedy-stte oxygen consumption (V O 2) during moderteintensity exercise (A) nd time-to-tsk filure during severe-intensity exercise (B), following consumption of 70, 140, nd 280 ml NO 3 -rich BR (gry brs) or NO 3 -depleted BR (PL; blck brs). End-exercise V O 2 during moderteintensity exercise ws reduced significntly following the ingestion of 280 ml BR. Time-to-tsk filure during severe-intensity exercise ws extended fter consumption of 140 ml BR with no further increse following 280 ml BR. Significnt difference from PL (P 0.05). lingul bcteri (16, 39). These phrmcokinetic responses to BR supplementtion re consistent with those reported previously following cute ingestion of KNO 3 (19). Together, these dt suggest tht the phrmcokinetics of plsm [NO 3 ] nd [NO 2 ] re dose dependent when NO 3 is dministered, either s NO 3 slt or in the form of nturl vegetble supplement. Ingestion of concentrted BR dose dependently lowered systolic BP nd MAP up to n intke of 8.4 mmol NO 3. More specificlly, cute ingestion of 4.2, 8.4, nd 16.8 mmol inorgnic NO 3, dministered in the form of BR, resulted in pek reductions of systolic BP of 5, 10, nd 9 mmhg nd pek reductions of MAP of 2, 5, nd 5 mmhg, respectively. Moreover, BR ingestion resulted in similr threshold effect on distolic BP, with pek reductions of 3 nd 4 mmhg following dministrtion of 8.4 nd 16.8 mmol NO 3 ; however, ingestion of 4.2 mmol NO 3 did not reduce distolic BP significntly. These reductions in BP re similr to those reported by Kpil et l. (19) following cute dministrtion of KNO 3, except tht Kpil et l. (19) reported dose-dependent reduction in BP up to 24 mmol KNO 3. The reson for this discrepncy between studies is uncler. Interestingly, compred with Kpil et l. (19), who reported 6 mmhg nd 9 mmhg reductions in systolic BP following the consumption of 12 mmol nd 24 mmol KNO 3, respectively, we observed lrger reductions in BP following the consumption of BR (e.g., pek reduction of 10 mmhg in systolic BP with 8.4 mmol NO 3 contined in 140 ml BR). It is possible tht this pprent greter potency of BR compred with NO 3 slt in reducing BP is relted to the polyphenols nd other ntioxidnts present in BR, which my fcilitte more efficient conversion of NO 3 to NO 2 (30). Interestingly, lthough the pek reduction in BP ws not significntly different between 8.4 nd 16.8 mmol NO 3, the men reduction in BP over 24 h ws dose dependent, with MAP, for exmple, reduced by 1, 2, nd 4 mmhg following dministrtion of 4.2, 8.4, nd 16.8 mmol NO 3, respectively. The results of the present study suggest tht BR (nd presumbly other NO 3 -rich vegetble) consumption cn provide nturl pproch to mintining or improving BP nd Tble 2. Hert rte nd blood lctte responses to moderte- nd severe-intensity exercise following supplementtion with 3 different volumes of beetroot juice nd plcebo 70 ml 140 ml 280 ml Plcebo Nitrte, 4.2 mmol Plcebo Nitrte, 8.4 mmol Plcebo Nitrte, 16.8 mmol Moderte-intensity exercise Hert rte, bets/min Bseline End-exercise Blood [lctte], mm Bseline End-exercise Severe-intensity exercise Hert rte, bets/min Bseline End-exercise Blood [lctte], mm Bseline Tsk filure Vlues re mens SD. [lctte], lctte concentrtion;, chnge. J Appl Physiol doi: /jpplphysiol

9 vsculr helth in young dults. The reductions in BP, evident in the present study, re noteworthy. For exmple, it hs been suggested tht lowering systolic BP by 10 mmhg my reduce the risk of ischemic hert disese by 25% nd the risk of stroke by 35% (27 29, 31). The beneficil hemodynmic effects of NO 3 supplementtion re thought to be due to the reduction of NO 3 to NO 2 nd then to NO within the blood vessel (13), resulting in rteril dilttion nd reduced peripherl resistnce (39). However, it is possible tht NO 2 itself my lso exert direct effect on the vsculr system, independent of NO formtion (1). There re severl dvntges to using inorgnic rther thn orgnic NO 3 for the prevention or tretment of hypertension (33). These include slow nd controlled increse in plsm [NO 2 ] following inorgnic NO 3 intke (due to NO 3 uptke into the enteroslivry circultion) compred with the more brupt chnges in plsm [NO 2 ] (perhps to toxic levels) nd BP, which cn occur with orgnic NO 3 dministrtion (33). Moreover, unlike the chronic dministrtion of orgnic NO 3,inorgnicNO 3 does not pper to led to the development of tolernce (37) nd endothelil dysfunction (33). S 2 : Dose Response The results of S 2 confirm tht concentrted BR consumption cuses dose-dependent increse in plsm [NO 3 ] by 334%, 778%, nd 1,556% nd plsm [NO 2 ] by 121%, 218%, nd 338%, 2.5 h postingestion of 4.2, 8.4, nd 16.8 mmol NO 3, respectively. The mgnitude of the increse in plsm [NO 2 ] following consumption of 8.4 nd 16.8 mmol NO 3 in the present study ws much lrger thn the pproximte % rise in plsm [NO 2 ], reported previously, following cute (pproximtely 4 6 mmol) (5, 21, 25, 37) nd chronic (pproximtely 5 6 mmol/dy) (2, 3, 22, 26, 37) dietry NO 3 supplementtion. This finding is likely consequence of the reltively higher NO 3 doses (8.4 nd 16.8 mmol NO 3 ) dministered in the present study. Interestingly, the group men plsm [NO 3 ] nd [NO 2 ] reported in S 2 re somewht lower thn those reported t 2 4 h postingestion of BR in S 1. Given tht there ws distinct subject recruitment for S 1 nd S 2, it is likely tht this discrepncy is due to individul vritions in the phrmcokinetic response to BR consumption. For exmple, when the individul plsm [NO 2 ] responses to the ingestion of 16.8 mmol NO 3 in S 1 re considered, pek concentrtions rnged from 493 to 1,523 nm, nd the time-to-pek concentrtion rnged from 130 to 367 min. The cuse of this wide interindividul vribility in the response of plsm [NO 2 ] to NO 3 ingestion is uncler, lthough it my depend, in prt, on slivry flow rte; lso, it is known tht the reduction of NO 3 to NO 2 is highly dependent on the ctivity of orl bcteri (16, 39). Another considertion is tht the bsolute NO 3 doses dministered in the present study (4.2, 8.4, nd 16.8 mmol in 1, 2, nd 4 BR shots, respectively) resulted in somewht different NO 3 doses when expressed reltive to BM ( , , nd mmol NO 3 /kg BM, respectively). Dose Response: Moderte-Intensity Exercise This is the first study to ssess the cute dose-dependent physiologicl responses to exercise following dietry NO 3 supplementtion in humns. We ssessed the cute response to three different doses of BR t 2.5 h postingestion, bsed on the Nitrte nd Exercise Dose Response Wylie LJ et l. significnt dose-dependent elevtion in plsm [NO 2 ] observed t 2 3 h postingestion in S 1 (Fig. 1B). The stedy-stte V O 2 mesured over the finl 30 s of moderte-intensity cycle exercise ws unffected by 4.2 mmol NO 3, tended to be lower (30 ml/min) following dministrtion of 8.4 mmol NO 3, nd ws reduced significntly (by 50 ml/min) following dministrtion of 16.8 mmol NO 3. The reduction in stedy-stte V O 2 (3%), observed following cute ingestion of 16.8 mmol NO 3 (0.23 mmol/kg BM), is similr to tht reported 2.5 h postingestion of 5.2 mmol NO 3 (0.07 mmol/kg BM) in the form of nonconcentrted BR (37) but is smller thn the 6% reduction reported 1 h postingestion of mmol/kg BM sodium nitrte (25). In contrst to cute ingestion, longer-term BR supplementtion (3 6 dys t pproximtely 5 7 mmol NO 3 /dy) resulted in n pproximte 5 7% reduction in stedy-stte V O 2 during moderte-intensity cycling (3, 26) nd running (22). Previous studies hve indicted tht the lowering of submximl exercise V O 2, following dietry NO 3 supplementtion, my result from improved mitochondril efficiency (25) nd/or reduction in the ATP cost of muscle force production (4). Altertions in protein expression hve been proposed s the mechnistic bsis for these effects (17, 24); however, it is unlikely tht these ltertions occur quickly enough to explin the effects observed so soon (1 2.5 h) fter NO 3 ingestion (25, 37). Alterntively, NO my cutely nd reversibly impct protein function through post-trnsltionl protein modifictions. For instnce, S-nitrostion of denine nucleotide trnslocse or other mitochondril or clcium-hndling proteins (35) my contribute to the cute reduction in O 2 cost of exercise following BR ingestion. The mechnistic bsis for the cute chnges in the O 2 cost of exercise following BR ingestion wrrnts further investigtion. An interesting observtion ws the dose-dependent increse in bseline nd end-exercise V CO 2, irrespective of condition (i.e., PL or BR). This smll but significnt rise in V CO 2 led to dose-dependent increse in RER tht ws more pronounced during bseline cycling compred with the exercising stedystte. An elevtion in RER is indictive of shift in substrte use towrd reltively greter relince on crbohydrte nd is likely due to the sugr content of the concentrted BR nd PL beverges (16 g/70 ml). Dose Response: Severe-Intensity Exercise J Appl Physiol doi: /jpplphysiol A novel finding of the present study ws tht 8.4 nd 16.8 mmol NO 3, but not 4.2 mmol NO 3, dministered cutely in the form of concentrted BR, significntly improved the timeto-tsk filure by 14% nd 12%, respectively, during severeintensity exercise. These findings re similr to the 14 16% improvement in exercise tolernce reported previously following 5 6 dys of BR supplementtion t lower dose (5 6 mmol NO 3 ) (3, 22). Although the mechnism(s) responsible for the ergogenic potentil of NO 3 supplementtion remin uncertin, they re believed to be medited vi biochemicl reduction of ingested NO 3 to biologiclly ctive NO 2 nd NO (4). NO hs been linked to the efficiency of erobic respirtion (9) nd the regultion of muscle contrction (35). Indeed, both more efficient mitochondril oxidtive phosphoryltion, vi reduced proton lek cross the inner mitochondril membrne