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Single and combined effects of beetroot juice and caffeine supplementation on cycling time trial performance

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Single and combined effects of beetroot juice and caffeine supplementation on cycling
time trial performance.
Stephen C. Lane1, John A. Hawley1,2, Ben Desbrow3, Andrew M Jones4, James R. Blackwell4, Megan L. Ross5, Adam J. Zemski5, Louise M. Burke5 Exercise & Nutrition Research Group, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia; 2 Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, United Kingdom; 3 School of Public Health and Griffith Health Institute, Griffith University, Gold Coast, QLD, Australia; 4 Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, St. Luke's Campus, Exeter, United Kingdom; 5 Sports Nutrition, Australian Institute of Sport, Belconnen, ACT 2626, Australia. Running Head: Nitrate and caffeine supplementation on cycling performance. Stephen Lane: [email protected]; Louise Burke: [email protected]; John A. Hawley: [email protected] Address for correspondence John A. Hawley: [email protected] Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 Exercise & Nutrition Research Group School of Medical Sciences Bundoora, Victoria 3083, AUSTRALIA For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 2 of 29
Abstract
Both caffeine and beetroot juice have ergogenic effects on endurance cycling performance. We investigated whether there is an additive effect of these supplements on the performance of a cycling time trial (TT) simulating the 2012 London Olympic Games course. Twelve male and 12 female competitive cyclists each completed four experimental trials in a double blinded Latin square design. Trials were undertaken with a caffeined gum (CAFF; 3 mg-1·kg-1 body mass [BM], 40 min prior to the TT), concentrated beetroot juice supplementation (BJ; 3 , 2 hours pre-TT), caffeine plus beetroot juice (CAFF+BJ) or a control trial (CONT). Subjects completed the TT (Females: 29.35 km; Males: 43.83 km) on a laboratory cycle ergometer under conditions of best practice nutrition: following a carbohydrate-rich pre-event meal; with the ingestion of a carbohydrate-electrolyte drink; and regular oral carbohydrate contact during the TT. Compared to CONT, power output was significantly enhanced after CAFF+BJ and CAFF (3.0% and 3.9% respectively, P < 0.01). There was no effect of BJ supplementation when used alone (-0.4%, P = 0.6; compared to CONT) or combined with caffeine (-0.9%, P = 0.4; compared CAFF). We conclude that caffeine (3 mg- ·kg-1 BM) administered in the form a caffeinated gum increased cycling TT performance lasting 50-60 min by 3-4% in both males and females. Beetroot juice supplementation was not ergogenic under the conditions of this study. Key Words: Cycling performance, nitrate, caffeine, ergogenic, time trial, carbohydrate Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 3 of 29
Athletes are continually striving to improve training capacity and performance. Not surprisingly, widespread use of a large number of nutritional supplements is commonplace in most sports as athletes search for a ‘magic bullet' that will elevate their performance to a higher level. Both caffeine (Desbrow et al. 2009; Irwin et al. 2011; Lane et al. 2013a) and 3 ) (Cermak et al. 2012a; Lansley et al. 2011a; Vanhatalo et al. 2011) have been shown to improve simulated road cycling performance in a variety of protocols. By mechanisms likely related to the central nervous system (CNS) (Costill et al. 1978; Tarnopolsky 2008) caffeine has been shown to improve arousal states (Backhouse et al. 2011) and reduce perceived exertion during steady state exercise (Backhouse et al. 2011; Doherty and Smith 2005; Lane et al. 2013a) resulting in enhanced performance during sustained high-intensity cycling events (Cox et al. 2002; Lane et al. 2013a; McNaughton et al. 2008). Accordingly, contemporary protocols for caffeine use are based on evidence that moderate intakes (3 mg-1·kg-1) of caffeine are equally as effective as larger doses (6 mg-1·kg-1) (Desbrow et al. 2012) for eliciting these CNS effects, and that caffeinated gums can also provide a rapidly absorbed caffeine dose (Kamimori et al. 2002; Ryan et al. 2013). With regard to dietary nitrate supplementation, Jones and co-workers (Bailey et al. 2010; Bailey et al. 2009; Lansley et al. 2011a; Lansley et al. 2011b; Vanhatalo et al. 2011) have reported that ingestion of beetroot juice increases exercise capacity through metabolic mechanisms that improve contraction efficiency within skeletal muscle. We hypothesised that the increased CNS drive and reduced perceived exertion elicited by caffeine supplementation in Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 combination with the previously reported improvements in metabolic efficiency resulting from beetroot juice ingestion would result in higher sustainable power outputs than when each supplement was taken in isolation. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 4 of 29
The specific aim of this project was to investigate the independent and combined effects of caffeine and NO - supplementation on the performance of a cycling task simulating the physical challenges of the London 2012 Olympic Games Road Cycling Time trial (TT). These effects were investigated against the background of a standardised dietary preparation including strategies that are typical of TT specialists; these included the intake of a small volume of fluid during the event and frequent mouth contact with carbohydrate (CHO) in the form of a sports confectionary, a practice recently confirmed as being beneficial to performance (Carter et al. 2004; Chambers et al. 2009; Lane et al. 2013b; Pottier et al. 2010), even when preceded by a CHO-rich pre-event meal (Lane et al. 2013b). We hypothesized that under optimal nutritional conditions i) caffeine alone and ii) NO - 3 alone supplementation would improve TT performance and iii) the concurrent use of caffeine and NO - supplementation would result in an additive performance enhancement than when each supplement was used in isolation. Twelve male [mean ± SD; age 31 ± 7, body mass (BM) 73.4 ± 6.8 kg, height 180.8 ± 6.1 cm, maximal aerobic power (MAP) 459.4 ± 31.1 W, peak oxygen consumption ( V & O2peak) 71.6 ± 4.6 ml·kg-1·min-1] and 12 female [age 28 ± 6, BM 62.1 ± 8.9 kg, height 169.1 ± 8.0 cm, MAP 327.1 ± 32.3 W, V & O2peak 59.9 ± 5.1 ml·kg-1·min-1] competitive cyclists or triathletes volunteered to participate in this study. Ethical clearance was obtained from the Australian Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 Institute of Sport Ethics committee. Prior to participation subjects were informed of the nature and risks involved and completed a medical questionnaire before providing written informed consent. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 5 of 29
Study overview On separate days following familiarisation (described subsequently), subjects performed four cycling time trials under different experimental conditions: Caffeine and beetroot juice supplementation (CAFF+BJ), caffeine and placebo beetroot juice (CAFF), beetroot juice and placebo caffeine (BJ) or a control trial consisting of a placebo of both caffeine and beetroot juice (CONT). All trials were separated by 7 days, and treatments were allocated using a double-blind Latin square design. Each ride was performed under standardised conditions representing optimal nutritional practice: CHO-rich ‘pre-event meal', ingestion of small amounts of a CHO-electrolyte drink during the TT, and regular oral CHO contact in the form of a sports confectionery product. All preliminary testing and experimental trials were performed under standard laboratory environmental conditions (STPD). Incremental cycle test In the 2 wk prior to their first experimental trial all subjects performed a progressive maximal exercise test to exhaustion on a cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands). After a 5 min warm up, the test protocol commenced at 175 and 125 W for males and females respectively and increased by 25 W every 60 s until volitional fatigue. Maximal aerobic power (MAP) was determined as the power output of the highest stage completed plus the fraction of any uncompleted workload as previously described (Ross et al. 2011; Ross et al. 2012). Expired gases were collected into a calibrated and customized Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 Douglas bag gas analysis system, which incorporated an automated piston that allowed the concentrations of O2 and CO2 (AEI Technologies, Pittsburg, PA) and volume of air displaced, to be quantified. The operation and calibration of this equipment have been described previously (Russell et al. 2002). Peak oxygen uptake (V & O2peak) was calculated as the highest average O2 consumption recorded over 60 s. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 6 of 29
Familiarisation session On the same day as the maximal test subjects completed a familiarisation ride on the same bike and simulated course they would complete in the subsequent experimental trials. In brief, subjects completed the course at their own self-selected intensity with the instruction to familiarize themselves with the course profile, bike set-up and the maximal intensity they believed they could sustain for the entire duration of the TT during subsequent rides. During this familiarization, dimensions for the bike set-up were recorded for replication throughout all experimental trials. Subjects were also familiarised with the use of the sports confectionery product (described subsequently) to be used during the experimental trials. Diet/Exercise control Subjects consumed a standardised diet for the 24 h period prior to each experimental trial using a pre-packaged standardised diet protocol described previously (Jeacocke and Burke 2010). Dietary goals for this period were 8 g-1·kg-1 BM CHO; 1.5 g-1·kg-1 BM protein; 1.5 g- ·kg-1 BM fat; and 220 kJ-1·kg-1 BM for the 24 h period. Subjects were instructed to avoid alcohol for the 24 h prior, and follow their habitual caffeine consumption patterns until 12 h prior to the start of the TT. Caffeine was not withheld for the 24 h period, since it has previously been shown a 3 mg-1·kg-1 BM dose of caffeine improves cycling performance irrespective of whether a withdrawal period is imposed on habitual caffeine users (Irwin et al. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 2011). The provided pre-trial standardised diets did not contain any NO - 3 rich products to avoid any possible effect on the experimental trials. A food menu was prepared for each subject based on individual BM and food preferences following an initial interview with a sports dietitian (AZ). During the same consultation For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 7 of 29
subjects reported the ongoing or acute use of any known medicine or supplement. In any case where the subject reported the use of a known medicine or supplement that may influence performance between trials, the subject was excluded from the study. The subjects' individual menu was prepared using Food Works Professional Edition, Version 6.0.2562 (Xyris Software, Brisbane, Australia). Subjects were provided with all foods and drinks in portion controlled packages for consumption during the first 22 h of the dietary control period, and were given verbal and written instructions on how to follow the diet. Checklists were used to record each menu item as it was consumed and to note any deviations from the menu. Each subject's food checklists were checked and clarified for compliance to the standardisation protocols by the sports dietitian prior to undertaking each trial. Analysis of the actual diet consumed by the subjects was undertaken on completion of the study using the same Experimental trials Subjects presented to the laboratory on four separate occasions each separated by 7 d. On each occasion subjects presented at the same time of day, voided their bladder prior to having their BM recorded then rested in a supine position for 10 min. At this time a Teflon canulla (Terumo, 20-22G, Tokyo, Japan) was inserted into a vein in the antecubital fossa. A resting blood sample (8 mL) was taken and the cannula flushed with saline to keep the vein patent for subsequent sampling. Two hours prior to the warm-up for each trial and immediately after the Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 resting blood sample subjects consumed the remainder of the control diet as a pre-"race" meal. This meal provided 2 g-1·kg-1 BM CHO which was included in the total CHO quota in the 24 h standardised diet. Subjects were instructed to consume their pre-"race" meal within 20 min, after which time they remained in the laboratory for the duration of that day's experimental trial. Depending upon the trial, either the experimental or placebo beetroot juice For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 8 of 29
concentrate was ingested in two separate doses (detailed below). Forty minutes prior to commencement of the TT subjects completed a standardised warm-up on the same bicycle as they performed the TT. The caffeine gum was administered in two doses, the first immediately prior to commencement of the warm up and the second immediately after its completion. Subjects then completed a TT simulating the characteristics of the London Olympic Games cycling TT course specific to the male or female events under the conditions as described below. Mean power output, heart rate and rating of perceived exertion were recorded during each trial. During the first trial water was provided ad libitum for the time period leading up to commencement of the TT. The volume consumed in this period was recorded and replicated throughout subsequent trials. The warm-up consisted of 30 min cycling at varying intensities (13 min at 25%, 5 min at 60%, 2 min at 70%, 3 min at 25%, 5 min at 60% and 2 min at 80% of MAP). Subjects then rested for 10 min prior to commencing the TT. Time Trials Subjects performed all experimental trials on a Velotron cycle ergometer (Racermate, Seattle, WA, USA) adjusted to the dimensions of their own bicycles. Males completed a simulated 43.83 km course while females completed a 29.35 km course. The courses were created using Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 global positioning satellite (GPS) data collected during a prior reconnaissance of the London Olympic TT event. Subjects were instructed to complete the TT as quickly as possible. Financial incentives were offered to encourage maximal effort. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 9 of 29
Experimental Interventions Beetroot Juice During two of the trials subject's received two separate doses of 140 mL of concentrated 3 rich beetroot juice delivering 8.4 mmol of NO3 in each dose (Beet it, James White Drinks Ltd., Ipswich, U.K.). Each subject ingested the first dose at a specific time 8-12 h prior to the commencement of each TT and was provided within each subjects controlled diet that was consumed the day prior to each experimental trial. The second dose was ingested in the laboratory 130 min prior to the commencement of the TT. During the two placebo trials, a similar tasting but NO - 3 depleted beetroot juice product ( 0.006 mmol of NO3 ; Beet it, James White Drinks Ltd., Ipswich) (Lansley et al. 2011b) was administered at identical time points as for the experimental trials. During the two caffeine trials a caffeinated gum (Stay Alert, Amurol Confectioners, Yorkville, IL, U.S.A.) was administered in 2 doses to deliver a total of 3 mg-1·kg-1 BM of caffeine. The gum was administered in a non-transparent package emptied directly into the mouth to avoid possible visual cues about the differences between trials (experimental vs. placebo). The first dose was administered immediately prior to the commencement of the warm up (40 mins prior to the TT) and consisted of a caffeine dose containing 2 mg-1·kg-1 BM. Subjects were instructed to chew the gum for a total of 10 min before it was removed Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 and discarded. The remaining dose containing 1 mg-1·kg-1 BM was administered under the same instructions at the end of the warm up (10 min prior to the TT). During the placebo trials, non-caffeinated gum matched for taste and texture (Jila Gum, Ferndale Confectionary Pty Ltd, Australia) was provided under the same conditions as the caffeinated gum. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 10 of 29
Carbohydrate Ingestion To ensure the findings of this study were relevant when applied in a ‘real world' situation in which athletes follow current nutritional guidelines to maximise performance, a carbohydrate sports gel (PowerBar Gel; Powerbar Inc, Florham Park, NJ) containing 28 g of CHO was ingested 15 min prior to the commencement of each TT. Additionally, at the commencement of each TT subjects were provided with a sports confectionary product (PowerBar Gel Blasts; Powerbar Inc, Florham Park, NJ, U.S.A.). Subjects were instructed to place the confectionery item in their mouth and leave it in their cheek cavity until it had completely dissolved, at which time another was provided. The timing and number of confectionery pieces used in the first trial was replicated throughout all subsequent trials. The aim of this procedure was to provide a constant CHO stimulus in the mouth similar to a CHO mouth rinse that has previously been shown to enhance cycling performance (Carter et al. 2004; Chambers et al. 2009; Fares and Kayser 2011; Lane et al. 2013b; Pottier et al. 2010). Subjects also received a CHO-electrolyte "sports drink" (Gatorade; Gatorade Co, Chicago, IL, U.S.A.) to consume at specific points during each TT. During the first trial males received two bottles, the first at 15 km and the second at 30 km during the TT whereas females received a single bottle at 15 km. These points correspond to portions of the TT in which prior reconnaissance of the course suggested it would be practical for competitors to take a drink. During the first trial, each bottle was pre-weighed and subjects instructed to consume as much fluid as desired within 1 min. Each bottle was then re-weighed and the volume of fluid consumed was recorded and Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 repeated throughout all subsequent trials. Blood collection and analysis At each sampling time point a total of 8 mL of whole blood was collected in a tube containing lithium heparin. Each trial included four sampling time points consisting of a For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 11 of 29
resting sample, one immediately prior to commencement of the warm up (prior to caffeine ingestion), a third immediately post the warm up and a final sample taken immediately post the TT. Tubes were immediately centrifuged at 4ºC at 4000 rev·min-1 for 10 min. The resultant plasma was divided into equal aliquots and stored at -80 ºC for the subsequent analysis of caffeine, NO - 3 and NO2 concentrations. Plasma caffeine concentration The quantitative analysis of plasma caffeine was performed using an automated "reverse phase" high-performance liquid chromatography system. Conditions were adapted with subtle modifications from Koch, Tusscher, Koppe and Guchelaar (Koch et al. 1999). The precise method has been described by us previously (Desbrow et al. 2009). Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 12 of 29
Plasma NO - 3 and NO2 concentration 3 and NO2 were analysed by gas phase chemiluminescence analysis. This initially required NO - 2 and NO3 to be reduced to nitric oxide (NO) gas. For reduction of 2 , undiluted plasma was injected into a glass purge vessel containing 5 mL glacial acetic acid and 1 mL NaI solution. For NO - 3 reduction, plasma samples were deproteinised in an aqueous solution of zinc sulphate (10% w/v) and 1M sodium hydroxide, prior to reduction to NO in a solution of vanadium (III) chloride in 1 M hydrochloric acid (0.8% w/v). Quantification of NO was enabled by the detection of light emitted during the production of nitrogen dioxide formed upon reaction of NO with ozone. Luminescence was detected by a thermoelectrically cooled, red-sensitive photomultiplier tube housed in a Sievers gas-phase chemiluminescence nitric oxide analyser (Sievers NOA 280i, Analytix Ltd, Durham, UK). The concentrations of NO - 2 and NO3 were determined by plotting signal area (mV) against a calibration plot of 25 nM to 1 µM sodium nitrite and 100 nM to 10 µM sodium nitrate respectively. Statistical Analysis Statistical analyses were performed using software package SPSS (Version 18). For all blood and physiological measures (combining both males and female results) one-way ANOVA's for repeated measures were used to compare between time points and trials using a Bonferroni adjustment where appropriate. Mean power output from the four trials were Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 analysed for males and females separately as well as combined using the magnitude based inference approach recommended for studies in sports medicine and exercise (Hopkins et al. 2009). The same inference based approach was also used to compare time to complete each trial for males and females separately. A spread sheet (Microsoft Excel), designed to examine post-only crossover trials, was used to determine the clinical significance of each treatment For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 13 of 29
(available at newstats.org/PostOnlyCrossover.xls), as based on guidelines outlined by Hopkins (Hopkins 2007). Qualitative inferences are reported as the percentage chance of a positive effect compared to the corresponding trial where a least worthwhile effect on power output of 1% was used as previously established (Paton and Hopkins 2006). Significance was set at P < 0.05. All data are presented as mean ±SD unless otherwise stated. Body mass There was no difference in BM upon presenting to the laboratory between trials. Similarly there was no statistical difference between trials for the change in BM pre- and post- each Plasma caffeine Figure 1A displays the plasma caffeine concentrations for all trials. At rest there was a small variation in plasma caffeine concentrations, likely because subjects were only instructed to abstain from caffeine in the 12 h prior to trials (Irwin et al. 2011). Within 30 min of ingestion plasma caffeine concentrations were significantly increased (CAFF+BJ 9.2 ±3.2, CAFF 10.0 ±3.80 µmol·L-1) compared to resting values and when compared to the non-caffeine trials. Peak caffeine concentrations (CAFF+BJ 16.7 ±3.1, CAFF 17.2 ±5.5 µmol·L-1) were recorded at the final collection point at the end of each TT. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 Plasma NO - 3 and NO2 Figure 1B shows plasma NO - 3 concentrations for all trials. The preloading NO3 dose administered 6-10 h prior to the resting blood sample increased plasma NO - 3 concentrations in the CAFF+BJ and BJ trials (113.1 ±33.3 and 123.2 ±37.6µmol·L-1 respectively; P < 0.01) For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 14 of 29
compared to the non-beetroot juice trials. Plasma NO - 3 levels remained significantly elevated in CAFF+BJ and BJ at all time points compared to CAFF and CONT (P < 0.05). The second dose of beetroot juice (administered 130 min prior to the TT) further elevated plasma NO - concentrations at 90 min (282.7 ±64.8 and 295.8 ±67.0 µmol·L-1) and 2 h post ingestion (310.6 ±58.7 and 333.9 ±64.7 µmol·L-1) compared to rest (P < 0.05) with concentrations remaining elevated until after the TT (334.1 ±53.3 and 343.1 ±58.4µmol·L-1; P < 0.05). Figure 1C shows plasma NO - 2 concentrations for all trials. Concentrations were significantly higher in CAFF+BJ and BJ at all time points compared to CAFF and CONT (P < 0.01). The resting blood sample reveals the preloading NO - 3 dose consumed 6-10 h prior to the resting blood sample elevated NO - 2 levels to 176.1 ±90.9 and 174.3 ±87.1 nmol·L-1 (P < 0.05) for the CAFF+BJ and BJ trials respectively compared to the non-beetroot juice trials. The second 3 rich beetroot juice did not elevate plasma NO2 concentrations further in the CAFF+BJ and BJ trials. Power output Figure 2 shows the relative mean power output combined for males and females. Caffeine improved mean power output compared to CONT in CAFF+BJ and CAFF trials on average by 3.5% (P <0.01). Beetroot juice supplementation had no effect on mean power output in either CAFF+BJ vs. CAFF or BJ vs. CONT. Using an inference based statistical approach Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 caffeine was very likely (99%) and very likely (97%) (CAFF+BJ vs. BJ and CAFF vs. CONT respectively) to have a positive effect on performance outcomes during a cycling TT. NO - supplementation was most unlikely (0%) and very unlikely (1%) (BJ vs. CONT and CAFF+BJ vs. CAFF respectively) to have any positive effect on performance. When mean power output was compared for trial order rather than intervention no significance was For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 15 of 29
detected between any trial (Trial 1 through 4; Males, 298 ±40, 301 ±35, 306 ± 40, 305 ±37 W respectively; Females, 212 ±30, 210 ±26, 212 ±34, 208 ±31 W respectively; P = 1.0) indicating the Latin square design was successful in eliminating any possible trial order Time trial completion time Times to complete the respective distances for males and females are presented in Table 1. For males when compared to CONT the time to complete the 43.83 km distance was reduced to a similar extent of 1.3% (P <0.05) for both the CAFF+BJ and CAFF trials. For females, the time to complete the 29.35 km distance was reduced by 0.9% and 1.6% (P <0.05) for the CAFF+BJ and CAFF trials respectively when compared to CONT. Beetroot juice supplementation had no significant positive or negative effect on time to complete the trials for both males and females in either CAFF+BJ vs. CAFF or BJ vs. CONT. Using an inference based statistical approach caffeine would possibly (65%) and likely (89%) for males and possibly (42%) and likely (88%) for females (CAFF+BJ vs BJ and CAFF vs. CONT respectively) produce a positive effect on performance outcomes during a cycling TT. 3 supplementation was unlikely (7%) and very unlikely (1%) for males and very unlikely (0%) under both conditions for females (CAFF+BJ vs CAFF and BJ vs. CONT respectively) to have any positive effect on performance. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 Heart rate and Rating of Perceived Exertion Table 1 shows mean heart rate and RPE for each trial for males and females. There were no differences in mean heart rate or RPE between any trials. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 16 of 29
Discussion
This is the first study to determine the single and combined effects of caffeine and NO - supplementation on the performance of cycling protocols that simulated real TT courses and were undertaken with the support of nutritional practices considered optimal for elite TT performance. As each of these ergogenic aids is purported to elicit their performance enhancing effect via different mechanisms (i.e., central vs. peripheral), we hypothesised that the combination of the two interventions would increase mean power output to a greater extent than when each intervention was administered in isolation. Our results indicate that caffeine supplementation provided a worthwhile enhancement of TT performance to both male and female cyclists, but that beetroot juice did not provide a detectable benefit under these conditions. In the current study, pre-event supplementation with caffeine (3 mg-1·kg-1 BM) increased mean power output in cycling time trials lasting 50 min (competitive female cyclists) and 60 min (competitive male cyclists) to a similar extent ( 3-4%) as reported previously using similar caffeine doses (Cox et al. 2002; Irwin et al. 2011; Jenkins et al. 2008; Lane et al. 2013a). In particular, these results are in agreement with the findings of Ryan et al. (2013) who reported that caffeine administered in the form of a gum prior to a cycling TT induced an elevation of circulating caffeine concentrations within 30 min of intake, and resulted in significantly improved performance. We observed that the benefits of ingesting caffeine 40 min prior to time trials simulating the specific courses undertaken at the 2012 London Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 Olympic Games were similar for males and females, although the courses they rode were slightly different in length and duration. Although these results were derived specifically for the preparation of cyclists for the 2012 Olympic Games, they can be generalised to other events of similar nature. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 17 of 29
It is worth commenting that caffeine ingestion improved performance in our study under standardised conditions of dietary preparation that are both recommended and typical of the practices of cycling TT specialists. These practices included a carbohydrate-rich pre-event meal (Lane et al. 2013b), consumption of a small fluid intake during the event according to the practical opportunities to drink (Garth and Burke 2013) and frequent mouth contact with carbohydrate (Lane et al. 2013b). Many studies often neglect to recognise that the real world application of ergogenic interventions may be influenced by optimal ‘race day' strategies. Indeed, a meta-analysis has shown that the benefits of caffeine ingestion on endurance performance are reduced when it is taken in combination with carbohydrate (Conger et al. 2011). However, under the conditions of our study caffeine ingestion still improved performance to the same degree as previously reported (Cox et al. 2002; Irwin et al. 2011; Jenkins et al. 2008; Lane et al. 2013a; Ryan et al. 2013) even when guidelines for optimal carbohydrate ingestion for this specific type of event (Burke et al. 2011) are followed. Lastly, our findings are also in agreement with Irwin et al. (2011) who reported a similar degree of improvement in cycling performance when a comparable 12 h withdrawal from caffeine was enforced in habitual caffeine users. This observation suggests that longer withdrawal periods (24- 48 h) as previously recommended (Burke 2008) may not be necessary. In contrast, we found no effect of beetroot juice ingestion on a cycling TT lasting 50-60 min Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 despite elevated plasma NO - 3 /NO2 concentrations. Indeed, the conditions under which supplementation with NO - 3 /beetroot juice ingestion enhances exercise capacity or performance remain somewhat unclear. Elements that could be of importance include the timing and dose of NO - 3 , the intensity and duration of the exercise protocol, and the training history or calibre of the athlete. Recently the effect of beetroot juice on exercise capacity has For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 18 of 29
been shown to be dose dependent, with the maximum benefits being seen with the acute ingestion of two bottles of beetroot juice concentrate (acute dose of 8.4 mmol NO - al. 2013). Since the cyclists in our study ingested the same amount of the same product, both acutely ( 2 h pre-exercise) and as an additional pre-load (6-10 h pre-trial), we are confident that our failure to detect benefits from NO - 3 supplementation cannot be explained by a sub- optimal dosing protocol. supplementation protocol substantially elevated plasma NO2 concentrations, although the peak values in our study were lower (225 vs. 470-687 nmol·L-1) than those reported by studies employing a similar acute dosing protocol in subjects with a range of training histories (Cermak et al. 2012b; Lansley et al. 2011a; Muggeridge et al. 2013; Wilkerson et al. 2012; Wylie et al. 2013). Although only speculative, it is possible that the pre-race meal, consumed shortly before the ingestion of the second beetroot juice dose, may have affected the conversion of NO - However, despite this observed difference Cermak et al. (2012b) and Wilkerson et al. (2012) reported significantly higher peak plasma 2 concentrations (532 and 472 nmol·L-1 respectively) compared to the current study, but also failed to detect a performance benefit in well-trained cyclists. Due to the range of plasma 2 concentrations, training histories as well as different performance tasks employed it is difficult to determine if these observations play a role in the effectiveness of NO - supplementation. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 The mechanism underpinning the observed benefits of NO - 3 supplementation on exercise capacity is believed to be a reduction in the oxygen cost of exercise, as a consequence of a reduced energy cost of contraction or enhanced mitochondrial efficiency (Jones et al. 2012). Whether this translates into an enhancement of performance across a range of exercise For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 19 of 29
intensities has not been systematically studied. However, it is worth noting that the performance of shorter cycling tasks ( 5-30 min duration) has been enhanced following NO - supplementation. For example, in a study by Lansley et al. (2011a), subjects who sustained intensities equivalent to 98% and 95% of VO2max during 4 km and 16.1 km time trials respectively recorded an improvement in performance after beetroot juice supplementation. However, a 50 mile cycling TT lasting 135 min and eliciting a sustained exercise intensity equivalent to 74% of VO 2max did not show a performance benefit following NO3 supplementation, despite subjects showing an improvement in power output per oxygen volume (W/L·min-1) (Wilkerson et al. 2012). Cermak et al. (2012b) also reported no enhancement of 1 h cycling TT performance in a cohort of well-trained cyclists using a similar acute NO - 3 dose and timing strategy as employed in the current study. Although we did not measure oxygen consumption during the TT in the current study, Coyle et al. (1991) reported that well-trained cyclists completed a 1 h TT at 87% VO2max, suggesting our comparable subjects worked at a lower percentage of their aerobic capacity than those observed in shorter duration tasks as employed in the study of Lansley et al. (2011a). Possible explanations for this observation include the effects of exercise intensity on muscle oxygenation and motor unit recruitment. Higher exercise intensities are likely to result in a greater degree of skeletal muscle hypoxia, which would be expected to facilitate NO production through the reduction of NO - 2 (Maher et al. 2008). In addition, higher exercise intensities would be expected to mandate a greater recruitment of type II muscle fibres. There Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 is evidence that the effects of NO - 3 supplementation on blood flow (Ferguson et al. 2013) and muscle force and calcium handling (Hernandez et al. 2012) might be more pronounced in type II fibres. These observations merit further investigation as it appears the effectiveness of 3 supplementation may be influenced by the intensity of the performance task. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 20 of 29
The failure to find a benefit of NO - 3 supplementation may be associated with the use of highly trained athletes due to a factor that is not currently identified. For example, studies in which pre-event ingestion of beetroot juice has been unable to produce a detectable improvement in performance have involved sub-elite or well-trained cohorts (Cermak et al. 2012b; Christensen et al. 2013; Muggeridge et al. 2013; Wilkerson et al. 2012). A recent meta-analysis of studies of beetroot juice/nitrate supplementation and endurance performance published before August 2012 found that the effects were more readily observed in inactive to recreationally active individuals (Hoon et al. 2013). Clearly, this intriguing aspect warrants further study, with candidate explanations including the optimisation of arginine-mediated pathways of NO production in highly-trained individuals or differences in muscle fibre-type (Christensen et al. 2013). It is noteworthy that Cermak et al. (2012a) reported a significant improvement in 10 km cycle TT performance following 6 days of beetroot juice supplementation but no effect on 1 hour TT performance after acute beetroot juice intake (Cermak et al. 2012b). While this might be related to differences in exercise duration and intensity, as discussed earlier, it is also possible that longer periods of beetroot juice supplementation are necessary for performance changes to be realised in highly-trained subjects. For example, changes in proteins related to mitochondrial efficiency (Larsen et al. 2011) and muscle calcium handling (Hernandez et al. 2012) that have been reported following nitrate supplementation are likely to take several Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 days (rather than hours) to become manifest. Previous studies have suggested that there may be "responders" and "non-responders" to 3 supplementation (Christensen et al. 2013; Wilkerson et al. 2012; Wylie et al. 2013) and this observation appears to be consistent within a highly trained cohort For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 21 of 29
(Christensen et al. 2013; Wilkerson et al. 2012). In the current study only two male individuals recorded better performances in both BJ vs CONT and CAFF+BJ vs. CAFF, suggesting they were possible "responders". In comparison Christensen and co-workers (2013) noted that two of the 10 highly trained cyclists in their study (mean aerobic capacity of 72.1 ml.kg-1.min-1 vs. 71.6 ml.kg-1.min-1 in the male cyclists in the current study) appeared to benefit from a chronic beetroot juice intake protocol, deriving a 3% improvement in performance of an 18 min TT compared with a control condition. Factors to explain individual responsiveness to such supplementation remain elusive at present. In conclusion we have provided evidence that a caffeine gum containing 3 mg-1·kg-1 BM ingested in the 40 min prior to a cycling TT lasting 45-60 min increases cycling power output in both males and females. However, despite increasing circulating NO - concentrations beetroot juice supplementation ingested 8-12 h prior as well as an acute dose ingested 2 h prior to the TT did not enhance cycling performance either in isolation or in combination with caffeine ingestion. Based on previous evidence that NO - 3 supplementation can improve performance under a variety of high intensity endurance tasks we cannot rule out the possibility that an additive effect may still be possible with different protocols or to specific individuals ("responders"). Further research is required to determine if NO - supplementation can further enhance performance when co-ingested with caffeine under shorter more intense tasks where the benefit of NO - 3 supplementation is more pronounced. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 22 of 29
We acknowledge the hard work and commitment of the athletes who gave their time to participate in this research project. We would also like to thank Greg Shaw for his assistance in the conduct of the study and the development of the placebo gum protocol as well as all of the people who assisted in recording data during the trials. This project was funded by a research grant from the Australian Institute of Sport (AIS) Sports Supplement Program and AIS Sports Nutrition. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 23 of 29
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Wylie, L.J., Kelly, J., Bailey, S.J., Blackwell, J.R., Skiba, P.F., Winyard, P.G., et al. 2013. Beetroot juice and exercise: pharmacodynamic and dose-response relationships. J Appl Figure 1: Plasma concentrations; A) caffeine, B) Nitrate (NO - 3 ), C) Nitrite (NO2 ), CAFF+BJ (beetroot juice with caffeine); CAFF (caffeine); BJ (beetroot juice); CONT (placebo of caffeine and beetroot juice) ($) different to CONT and BJ (P < 0.01); (a) different to REST and 0 min (P < 0.01); (b) different to REST, 0 min and 30 min (P < 0.05); (c) different to Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 REST; (γ) different to CONT and CAFF (P < 0.01); Times relative to ingestion; Rest in B and C include a ‘preload' NO - 3 dose 6-10 h prior; Mean ± SD. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 26 of 29
Figure 2: Mean power output combined for males and females; CAFF+BJ (beetroot juice with caffeine); CAFF (caffeine); BJ (beetroot juice); CONT (placebo of caffeine and beetroot juice); (*) different to CONT and BJ (P < 0.01); Mean ± SD. Table 1: Summary of cycling time trial performance and associated measures. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 27 of 29
Heart Rate
Body Mass
1:03:30.39 ± 0:03:16.15 1:02:38.04 ± 0:03:31.00 * 314 ± 44 * 68.1 ± 6.3 * 1:02:43.86 ± 0:03:04.87 * 313 ± 38 * 67.8 ± 4.5 * 1:04:05.03 ± 0:02:50.09 0:51:40.10 ± 0:02:31.71 0:51:11.88 ± 0:02:22.13 * 212 ± 27 * 64.9 ± 5.1 * 0:50:50.53 ± 0:02:56.48 * 0:51:41.06 ± 0:02:39.51 66 ± 5.6 * CAFF+BJ vs CONT 3.1 ± 1.9 66.6 ± 5.4 * BJ vs CONT * Significanlty different to CONT and BJ (P < 0.05) Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

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Figure 1: Plasma concentrations; A) caffeine, B) Nitrate (NO3-), C) Nitrite (NO2-), CAFF+BJ (beetroot juice with caffeine); CAFF (caffeine); BJ (beetroot juice); CONT (placebo of caffeine and beetroot juice) ($) Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 different to CONT and BJ (P < 0.01); (a) different to REST and 0 min (P < 0.01); (b) different to REST, 0 min and 30 min (P < 0.05); (c) different to REST; (γ) different to CONT and CAFF (P < 0.01); Times relative to ingestion; Rest in B and C include a ‘preload' NO3- dose 6-10 h prior; Mean ± SD. For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

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Figure 2: Mean power output combined for males and females; CAFF+BJ (beetroot juice with caffeine); CAFF (caffeine); BJ (beetroot juice); CONT (placebo of caffeine and beetroot juice); (*) different to CONT and BJ (P < 0.01); Mean ± SD. Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by CAULBF on 12/12/13 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

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