Effects of Dietary Nitrate, Caffeine, and Their Combination on 20-km Cycling Time Trial Performance

Abstract Glaister, M, Pattison, JR, Muniz-Pumares, D, Patterson, SD, and Foley, P. Effects of dietary nitrate, caffeine, and their combination on 20-km cycling time trial performance. J Strength Cond Res 29(1): 165–174, 2015—The aim of this study was to examine the acute supplementation effects of dietary nitrate, caffeine, and their combination on 20-km cycling time trial performance. Using a randomized, counterbalanced, double-blind Latin-square design, 14 competitive female cyclists (age: 31 ± 7 years; height: 1.69 ± 0.07 m; body mass: 61.6 ± 6.0 kg) completed four 20-km time trials on a racing bicycle fitted to a turbo trainer. Approximately 2.5 hours before each trial, subjects consumed a 70-ml dose of concentrated beetroot juice containing either 0.45 g of dietary nitrate or with the nitrate content removed (placebo). One hour before each trial, subjects consumed a capsule containing either 5 mg·kg−1 of caffeine or maltodextrin (placebo). There was a significant effect of supplementation on power output (p = 0.001), with post hoc tests revealing higher power outputs in caffeine (205 ± 21 W) vs. nitrate (194 ± 22 W) and placebo (194 ± 25 W) trials only. Caffeine-induced improvements in power output corresponded with significantly higher measures of heart rate (caffeine: 166 ± 12 b·min−1 vs. placebo: 159 ± 15 b·min−1; p = 0.02), blood lactate (caffeine: 6.54 ± 2.40 mmol·L−1 vs. placebo: 4.50 ± 2.11 mmol·L−1; p < 0.001), and respiratory exchange ratio (caffeine: 0.95 ± 0.04 vs. placebo: 0.91 ± 0.05; p = 0.03). There were no effects (p ≥ 0.05) of supplementation on cycling cadence, rating of perceived exertion, , or integrated electromyographic activity. The results of this study support the well-established beneficial effects of caffeine supplementation on endurance performance. In contrast, acute supplementation with dietary nitrate seems to have no effect on endurance performance and adds nothing to the benefits afforded by caffeine supplementation.


INTRODUCTION
N itrate supplementation, through natural (beetroot juice) and pharmacological (nitrate salts) methods, is an emerging area of research because of its potential to improve endurance performance. Many, although not all (4,5,13,36), studies have reported a reduction in the oxygen cost of exercise after nitrate supplementation (1,2,11,(28)(29)(30)33). Correspondingly, several studies have reported improvements in time-toexhaustion (1,2,28) and time-trial (6,11,27) protocols, with improvements in the latter of 1.2-2.8% in events lasting from 6 to 27 minutes (11,27). Mechanisms to explain these performance enhancements, although unresolved, are suggested to result from a bioconversion cascade from nitrate, through nitrite, to nitric oxide leading to improvements in mechanical efficiency (1), mitochondrial-coupling efficiency (29), or both. As such, and despite several contradictory findings (4,5,12,13,31,33,36,43), acute nitrate supplementation in the form of concentrated beetroot juice is currently marketed to athletes as a means of enhancing endurance performance.
In contrast to the above, the effects of caffeine supplementation on endurance performance are more clearly defined (10, 20), with doses of 3-6 mg$kg 21 producing positive effects (1.2-4.2%) in time trial events lasting 5-60 minutes (8,9,26,32,39,42). Although the effects of caffeine were originally purported to emanate from a glycogen-sparing mechanism of action, the absence of a corroborative change in respiratory exchange ratio (RER), combined with the evidence of significant effects in events where glycogen availability would not be a limiting factor (20), led researchers to consider alternative explanations. At present, although a small peripheral effect through enhanced calcium mobilization remains a possibility (40), the key mechanism by which caffeine is believed to enhance endurance performance is through a central mechanism involving an antagonism of adenosine receptors and leading to increases in neurotransmitter release, motor unit firing rates, and pain suppression (25), with particular support for the latter (40). Given the apparent differences in their mechanisms of action (peripheral [nitrate] vs. central [caffeine]) and the fact that caffeine has been shown to have no scavenging effects on nitric oxide, at least in rodents (38), it is possible that both the supplements combined could have a greater effect on endurance performance than either supplement alone. The aim of this study was therefore to examine the acute supplementation effects of dietary nitrate, caffeine, and their combination on subsequent 20-km time trial performance in well-trained athletes.

Experimental Approach to the Problem
In this investigation, subjects were required to complete 6 trials consisting of a familiarization trial (trial 1), a stepwise incremental trial (trial 2), and 4 supplementation trials (trials 3-6). The stepwise incremental trial was used to provide descriptive data and to enable time trial performance to be evaluated relative to standard physiological parameters. The supplementation trials followed a Latinsquare design and were double blind, randomized, and counterbalanced. Subjects were instructed to maintain their normal diet throughout the testing period, to follow the same diet for 24 hours before each trial, to avoid food and drink in the hour before each trial, and to refrain from strenuous exercise for 24 hours before each trial. Subjects were provided with lists of caffeine-and nitrate-rich foods and instructed to abstain from consumption of these for 24 and 48 hours, respectively, before each trial. A questionnaire was used to investigate the possible effect that normal caffeine consumption may have on the results of the investigation. Subjects were provided with low-nitrate bottled drinking water (Buxton Still; NestléWaters UK, Ltd., Buxton, United Kingdom) to assist with adherence to the dietary restrictions. In addition, subjects were instructed to refrain from using antibacterial mouthwash for 48 hours before each trial to avoid disruption of nitrate-reducing bacteria in the enterosalivary circulation (19). Approximately 2.5 hours before each of the supplementation trials, subjects consumed a 70-ml dose of concentrated beetroot juice (Beet IT Sport Shot; James White Drinks, Ltd., Suffolk, United Kingdom) containing either 0.45 g (;7.3 mmol) of dietary nitrate, or, as supplied specifically by the manufacturer for research purposes, with the nitrate content removed (placebo: ;0.01 mmol nitrate). One hour before each trial, subjects consumed a gelatin capsule containing either 5 mg$kg 21 of caffeine (Sigma-Aldrich, Steinheim, Germany) or placebo (maltodextrin; My Protein, Manchester, United Kingdom). The supplementation trials therefore consisted of 4 conditions: placebo (placebo + placebo), nitrate (nitrate + placebo), caffeine (caffeine + placebo), and caffeine + nitrate.

Subjects
Fourteen well-trained, competitive, female athletes (cyclists and triathletes) volunteered for the study, which was approved by the St. Mary's University Ethics Committee. Before testing, subjects received written and verbal instructions regarding the nature of the investigation and completed a training history questionnaire, which indicated that all had been actively involved in sports activities for approximately 13 years and that, at the time of the investigation, time spent training each week was 10.7 6 2.2 hours. Before commencement, all subjects completed a health-screening questionnaire and provided written informed consent. Mean 6 SD for age, height, body mass, and body fat of the subjects were 31 6 7 years, 1.69 6 0.07 m, 61.6 6 6.0 kg, and 24.9 6 4.3%, respectively.

Procedures
All trials were performed at approximately the same time of day (61 hour) in an air-conditioned laboratory maintained at a temperature of 198 C. In all trials, subjects exercised on a racing bicycle (Claud Butler San Remo; Claud Butler, Brigg, United Kingdom) seated on a motor-braked turbo trainer (Tacx Fortius, Wassenar, the Netherlands), which has been shown to have very good test-retest reliability (coefficient of variation: 1.6%) for 20-km time trial performance (37). The bicycle was fitted with clipless pedals and the subjects cycled using their own cycling shoes. Rear tyre pressure was maintained at 100 psi and the trainer was calibrated before each trial in accordance with the manufacturer's instructions. Before every familiarization trial, the saddle height was adjusted for each subject and noted for future replication.
Each stepwise incremental trial began at 100 W and increased by 20 W until blood lactate was .4 mmol$L 21 . The duration of each increment was 3.5 minutes, and a 20-mL capillary blood sample was obtained in the last 30 seconds of each increment for the evaluation of blood lactate through an automated analyzer (Biosen C-Line; EKF Diagnostic, Ebendorfer Chaussee, Barleben, Germany). After a 5-minute passive rest period, subjects completed a second incremental test, again starting at 100 W and increasing by 20-W increments; however, for this phase of the trial, the duration of each increment was only 1 minute. The trial was terminated when subjects reached volitional exhaustion, at which time a final blood lactate measurement was obtained. Oxygen uptake (V _ O 2 ) was monitored (breath by breath) throughout using an online gas analyzer (Oxycon Pro; Jaeger, Hoechberg, Germany). The analyzer was calibrated before each trial using oxygen and carbon dioxide gases of known concentrations (Cryoservice, Worcester, United Kingdom), and the flowmeter was calibrated using a 3-L syringe (Viasys Healthcare GmbH, Hoechberg, Germany). During the trials, subjects breathed room air through a facemask (Hans Rudolph, Kansas City, MO, USA) that was secured in place by a head-cap assembly (Hans Rudolph). Maximal oxygen consumption was determined as the highest 30-second average V _ O 2 recorded during the trial provided that at least 2 of the Dietary Nitrate and Caffeine on Cycling following criteria had been met: (a) a plateau in V _ O 2 , as determined by an increase of less than 2 ml$kg 21 $min 21 over the previous stage, (b) a RER $1.15, (c) a heart rate within 10 b$min 21 of age-predicted maximum, and (d) a blood lactate concentration $8 mmol$L 21 .
The familiarization and supplementation trials began with subjects completing a 5-minute warm-up at 100 W, followed by a 5-minute period of passive rest. Subjects then completed a 20-km time trial against a resistance designed to replicate outdoor, level-gradient cycling conditions. All measures of elapsed time were removed from the testing environment, and the only data visible to the subjects throughout each time trial were the distance completed. Verbal encouragement was provided throughout the trial. Subjects were free to change gears throughout familiarization; however, the gearing and cadence typically chosen were noted and used to standardize subsequent warm-up performance and the starting intensity for subsequent time trials. After the start of each supplementation time trial, subjects were free to change gears if they wished. Power output, distance completed, and cadence were recorded at 1 Hz throughout each time trial. Expired air was monitored, breath-by-breath,  for the evaluation of V _ O 2 and RER. Heart rate was monitored at 5-second intervals using a heart rate monitor (Polar s610; Polar Electro Oy, Kempele, Finland). Rating of perceived exertion (RPE) values were recorded at 5-km intervals using a 15-point scale (7).
Five minutes before the start of each supplementation trial, a venous blood sample was drawn from a branch of the basilic vein, collected in lithium-heparin tubes (Vacutainer; Becton Dickinson, Oxford, United Kingdom), mixed, and immediately centrifuged 1,600 g at 3,000 rpm for 10 minutes at 48 C. Subsequently decanted plasma samples were frozen at 2808 C until analyzed for nitrate/nitrite and caffeine content using chemiluminescence and high-performance liquid chromatography, respectively. Analysis of plasma nitrate and nitrite content through chemiluminescence was performed using the same procedures outlined by Peacock et al. (36).
Before the supplementation trials, subjects lay supine on an inclined couch while pre-gelled disposable hypoallergenic 1-cm snap-electrodes (Performance Plus; Vermed, Bellows Falls, VT, USA) were located over the belly of the vastus lateralis of the right leg for the evaluation of muscle activity using integrated electromyography (iEMG). Positioning of the electrodes was made using SENIAM (Surface Electromyography for the Non-Invasive Assessment of Muscles) guidelines. Skin surfaces were shaved, if necessary, and swabbed with alcohol before electrode placement. Electrodes were placed 2.5 cm apart, parallel to the direction of muscle fibers, with a reference electrode located above the tibia. The position of the electrodes was outlined with indelible pen to replicate electrode placement in subsequent trials. Electromyography data were sampled at 1,000 Hz using a data acquisition system (Biopac MP150; Biopac Systems, Inc., Goleta, CA, USA). Data were sampled for 30 seconds midway through each warm-up and in the final minute before the end of each 5-km interval during each time trial. The raw data was band-pass filtered (10-500 Hz), rectified, and integrated over each 30-second time period. Integrated electromyography data from each time trial were normalized to the warm-up data to allow for any subtle movements in electrode placement between trials.
The influence of supplementation on tissue oxygenation was evaluated using a continuous wave near-infrared spectroscopy (NIRS) system (PortaMon; Artinis Medical Systems, Zetten, the Netherlands), which uses 2 wavelengths of light (842 and 762 nm) to measure concentration changes in oxyhemoglobin ([HbO 2 ]) and deoxyhemoglobin ([HHb]), and providing an index of tissue saturation. The midpoint between the light source (optode) and the receiver was located over the belly of the left vastus lateralis using the same procedure for electrode placement outlined above, with the device aligned parallel to the direction of the muscle fibers. Before identifying the location, the area beneath the device was shaved, if necessary, swabbed with alcohol, and covered with a 6 3 7-cm transparent adhesive dressing (Tegaderm; 3M, St. Paul, MN,

Statistical Analyses
All statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS for Windows; SPSS, Inc., Chicago, IL, USA). Measures of centrality and spread are presented as mean 6 SD values. Oxygen uptake data from each time trial were filtered to eliminate values that were outside 4 SDs of the midpoint of a rolling 20 breath mean (attributed to "noise"), integrated, and averaged to provide mean responses for each trial and each 5-km split. The onset of blood lactate accumulation (OBLA), from the incremental trials, was identified using software developed for the purpose (34). The effects of supplementation on pretest measures of plasma nitrate, nitrite, and caffeine were evaluated using 1-way analyses of variance (ANOVAs). Two-way (supplement 3 5-km split) ANOVAs were used to determine the effects of supplementation and 5-km splits on the 20-km time trial performance measures (power output, time, and cadence) and physiological responses (heart rate, blood lactate, V _ O 2 , RPE, and RER). The possibility that any effects of caffeine supplementation on performance were influenced by habitual caffeine consumption was investigated by deriving correlations between estimated daily caffeine consumption and caffeine-induced changes (relative to placebo) in 20-km time trial performance. The effects of supplementation on iEMG activity during the warm-up and each time trial were evaluated using 1-and 2-way (supplement 3 5 km split) ANOVAs, respectively. Finally, the effects of supplementation on NIRS data at rest, during warm-up, and during each time trial were determined using 1-way ANOVAs. For all analyses, a was set at 0.05. Violations to assumptions of sphericity were adjusted using the Greenhouse-Geisser correction factor. Significant interactions were followed-up using post hoc tests with Bonferroni adjustments for multiple comparisons. The above analyses provided 95% confidence limits for all estimates.

Plasma Analyses
The results of the plasma nitrate, nitrite, and caffeine analyses are presented in Table 1
Respiratory Exchange Ratio. The pattern of the RER response to the time trials is presented in Figure 1C. There was a significant effect of 5-km split (F 1.2,15.9 = 23.51; p , 0.001) on RER, with significant differences between all comparisons apart from that between the 5-10 and 15-20 km splits. There was also a significant effect of supplementation (F 3,39 = 6.61; p = 0.001), with post hoc tests revealing significantly higher RER values for caffeine vs. placebo (mean difference: 0.033; 95% likely range: 0.002-0.063) and caffeine vs. nitrate (mean difference: 0.034; 95% likely range: 0.001-0.064) trials only. There was no significant supplement 3 split interaction (F 4.0,52.0 = 1.08; p = 0.38) for RER.
Ratings of Perceived Exertion. There was a significant effect of 5-km split on RPE during the time trials (F 1.6,21.3 = 144.75; p , 0.001). Post hoc comparisons revealed a progressive increase in RPE throughout the time trials with significant differences between all contrasts (Table 3). There was, however, no signif-icant effect of supplementation (F 3,39 = 1.62; p = 0.20) and no supplement 3 split interaction (F 9,117 = 0.82; p = 0.60).
Blood Lactate. Blood lactate responses to the time trials are presented in Figure 1D. There was a significant effect of 5-km split (F 1.6,20.7 = 23.94; p , 0.001), with mean values increasing throughout the time trials and with post hoc analyses revealing significant differences between all contrasts apart from those between 0-5 and 5-10 km, and 5-10 and 10-15 km splits. There was also a significant effect of supplementation (F 2.0, 26 , and caffeine + nitrate vs. placebo (mean difference: 2.50 mmol$L 21 ; 95% likely range: 1.14-3.85 mmol$L 21 ). The supplement 3 split interaction was not significant (F 9,117 = 1.94; p = 0.053).
Tissue Oxygenation. The effects of supplementation on NIRSderived measures of tissue oxygenation at rest, during warmup, and during the time trials are presented in Table 4. There were no significant effects of supplementation on any of the measures apart from [HHb] (F 3,39 = 5.34; p = 0.004) during the time trials. Post hoc analyses revealed significantly lower values for [HHb] in the caffeine + nitrate vs. the nitrate trial only (mean difference: 3.90; 95% likely range: 0.84-6.96).

DISCUSSION
The aim of this study was to examine the acute supplementation effects of dietary nitrate, caffeine, and their combination  Journal of Strength and Conditioning Research the on subsequent 20-km time trial performance in well-trained competitive athletes. Relative to placebo, the results showed a significant effect of caffeine, but no effect of nitrate. Moreover, although performance after caffeine + nitrate was not significantly different from the caffeine only condition, it was not significantly different from the nitrate and placebo conditions.
The pretrial plasma nitrate and nitrite concentrations observed in the caffeine and placebo conditions are commensurate with values previously reported for the same level of dietary nitrate restriction (30). Moreover, the increases in plasma nitrate and nitrite concentrations, relative to placebo, are consistent with those reported after similar acute nitrate dosing strategies (12,28). Nevertheless, nitrate supplementation had no effect on time trial performance. Apart from the findings of Lansley et al. (28), and the final three of six 500-m rowing repetitions (no overall effect) used by Bond et al. (6), previous research, using various modes of exercise and time trial durations (4.5-138 minutes), have also found no significant effect of acute nitrate supplementation on endurance performance (12,13,33,36,43). Since the nitrate dosing strategies used by Lansley et al. (28) and Bond et al. (6) fall within the range used by the aforementioned investigations and since all the studies listed used well-trained subjects, it is difficult to provide an explanation for these discrepancies.
Previous research has generally, but not always (4,5,13,36), reported a reduced oxygen cost of submaximal exercise after acute or chronic nitrate supplementation (1,2,11,27,29,30,33). In those studies that have examined time trial performance, several have failed to evaluate performance relative to V _ O 2 (6,(11)(12)(13)36). Of those that have, Muggeridge et al. (33) reported a reduction in V _ O 2 , despite no change in performance; Lansley et al. (28) found no change in V _ O 2 , despite an increase in performance; and Wilkerson et al. (43), despite finding no change in V _ O 2 or performance, found an increase in power output relative to V _ O 2 . In this study, subjects completed the nitrate-supplemented time trials at a mean V _ O 2 of approximately 83% of V _ O 2 max (;95% of OBLA). As such, it is possible that the absence of an effect of nitrate on V _ O 2 could be due to the intensity of the protocol. Indeed, Larsen et al. (30) found that nitrate supplementation only reduced V _ O 2 at submaximal intensities of #80 V _ O 2 max. Of course, it is also possible that the nitrate dose delivered was insufficient to produce an effect. However, because plasma nitrate and nitrite concentrations were significantly elevated, relative to placebo, before each time trial, and because the nitrate dose administered was only slightly less (;1 mmol) than the dose recently reported to optimize the effect of acute nitrate supplementation on submaximal V _ O 2 and a time-to-exhaustion task (44), this suggestion seems unlikely.
As with nitrate, postsupplementation plasma caffeine concentrations were similar to values previously reported for the same dosing strategy (18). Moreover, the effect of caffeine supplementation on time trial performance adds to the considerable body of previous research supporting a positive effect of caffeine on endurance exercise (10, 20). Although research into the effects of caffeine on time trial performance, particularly in well-trained athletes, is less substantive; it generally, but not always (14,22), corroborates the results of this study (8,9,32,39,42). Nevertheless, the increase in power output was not accompanied by a change in cadence, suggesting that the effect was because of an increase in force production rather than an increase in the frequency of each pedal stroke. However, this response was not reflected in an increase in iEMG activity. Because the key mechanism by which caffeine is believed to enhance exercise performance is through the antagonism of adenosine receptors, leading to increases in neural drive and pain suppression (25), the absence of an effect on iEMG suggests that the procedure may lack the necessary sensitivity to detect changes of the magnitude observed in this study.
Although differences in protocol design make direct comparisons difficult, many of the physiological responses that accompanied caffeine supplementation are consistent across studies. For instance, caffeine has consistently been shown to alter perceptual responses either, as in this study, by allowing a greater amount of work to be performed at the same perception of effort or by reducing the perception of effort for the same exercise intensity (16). Similarly, several studies that have observed a caffeine-induced increase in performance have, as in this study, also reported a corresponding elevation in heart rate (8,26,39). Although the suppressive effect of caffeine on RPE is most likely because of caffeine's antinociceptive effects (25), the increase in heart rate is most likely reflective of the caffeine-induced increase in power output coupled with a progressive increase in cardiovascular drift. Although it is also possible that caffeine exerted a direct effect on heart rate, research into the effects of caffeine on fixed-intensity submaximal exercise generally shows no effect (24,26,39).
Given the normally strong positive association between heart rate and V _ O 2 at submaximal intensities, it was surprising that the significant effect of caffeine on heart rate was not reflected in a corresponding effect on V _ O 2 . However, caffeine did have a significant effect on blood lactate, suggesting that the caffeine-induced increase in time trial performance was because of an increased contribution from anaerobic metabolism. Although this suggestion provides a fitting explanation for the above responses, Graham et al. (21) could not attribute caffeine-induced increases in blood lactate to an increase in lactate release from working muscles. Although further research is required to resolve this issue, the caffeineinduced increase in blood lactate, in the absence of any change in V _ O 2 , provides an explanation for the observed increased in RER through an increased buffering of associated hydrogen ions; however, it is difficult to resolve why this effect was not observed in the caffeine + nitrate trial or in other performance-based studies (9,24).
Typical daily caffeine consumption of the athletes in this study was similar to that of the general population Dietary Nitrate and Caffeine on Cycling (.200 mg$d 21 ) (17). Although there is some evidence that caffeine habituation may reduce the effects of supplementation (3), the fact that the relationship between habitual daily caffeine consumption and the caffeine-induced increase in power output was small adds support to most studies that report findings to the contrary (15,18,41).
Despite the significant effect of caffeine on time trial performance, the absence of a significant effect when caffeine was combined with dietary nitrate suggests a possible negative interaction between the supplements. Indeed, the lack of a significant difference in plasma nitrite concentration between the caffeine + nitrate and the caffeine conditions adds some credence to this idea. However, the effects on time trial performance were similar between caffeine and caffeine + nitrate conditions; moreover, relative to placebo, heart rate and blood lactate responses for the combined condition were similar to those of caffeine, suggesting that the magnitude of this effect is small. Considering the substantial differences between the timings of nitrate and caffeine administration, it seems unlikely that any negative interactive effects occurred before absorption; however, because possible interactions between caffeine and nitrate in blood do not appear previously to have been investigated, further research is needed to confirm and explain this finding.
Although the caffeine + nitrate condition failed to significantly enhance time trial performance, it did, relative to the nitrate condition, result in a significant reduction in [HHb]. It is difficult to reconcile this effect, particularly given the lack of a corresponding significant effect with caffeine and the absence of a difference in [HHb] between the caffeine + nitrate and the placebo conditions. Bailey et al. (2) observed a significant reduction in [HHb] amplitude during moderateintensity exercise coupled with an increase in [HbO 2 ] at rest and during moderate-intensity exercise after chronic (6.2 mmol$d 21 for 6 days) nitrate supplementation. In contrast, the results obtained at rest and during warm-up in this study, albeit using an acute dosing strategy, failed to show the same responses. However, Bailey et al. (2) observed no effect of nitrate on NIRS-derived indices of muscle oxygenation during "severe" exercise, again supporting the idea that the lack of an effect of nitrate in most time trial studies (12,13,33,36,43) is because of the intensity of the protocols.
This study used competitive female athletes; first, because they were well-trained and secondly, because there is a lack of research in this population. The busy training schedules of the athletes, combined with the need to allow for sufficient recovery between trials, meant that possible circa-mensal effects could not be controlled. However, although there is some evidence to the contrary, and despite several methodological issues and a lack of research using well-trained athletes, many studies have found no effect of the menstrual cycle on cardiorespiratory measures or exercise performance (23,35). As such, and given the randomized order of supplement administration, this was not considered to be a major limitation to the investigation.

PRACTICAL APPLICATIONS
The results of this study support the well-established beneficial effects of caffeine supplementation on endurance performance. In contrast, acute supplementation with dietary nitrate, in the form of a commercially available sport-specific beetroot supplement, had no effect on 20-km cycling time trial performance or any associated physiological responses. For those athletes who may be inclined to take combinations of supplements to enhance endurance performance, the results of this study suggest that acute supplementation with dietary nitrate adds nothing to the ergogenic benefits of caffeine supplementation and, although further research is required to confirm, may even have an antagonistic effect.