Acute beetroot juice supplementation did not enhance intermittent running performance in trained rugby players

ABSTRACT
 Purpose: Since the effect of dietary nitrate (NO3 -) supplementation on rugby performance is unclear, the aim of the present study was to determine the effect of acute NO3 - supplementation, on the modified Yo-Yo intermittent recovery level 1 (IR1) performance test in trained male rugby players. Methods: In a randomised, counterbalanced, double-blind, placebo-controlled crossover design, 12 trained rugby union players performed two experimental trials three hours after supplementation of either 140 mL NO3 --rich (BRJ; ∼12.8 mmol NO3 -) or NO3 --depleted (PLA) BRJ. After blood sampling, players performed the modified Yo-Yo IR1 test. Countermovement jumps (CMJ) were also measured before (pre-CMJ) and after (post-CMJ) the prone Yo-Yo IR1 test. Results: Plasma NO3 - (BRJ: 570 ± 146 µM vs. PLA: 72 ± 23 µM) and nitrite (NO2 -) concentrations (BRJ: 320 ± 123 nM vs. PLA: 103 ± 57 nM) were increased after BRJ compared to PLA supplementation (both P < 0.001). Performance in the modified Yo-Yo IR1 test did not differ between BRJ (542 ± 209 m) and PLA (498 ± 185 m, P = 0.3). The jump height in pre-CMJ and in post-CMJ were similar between trials (both P > 0.05). Conclusions: Acute BRJ supplementation increased plasma NO3 - and NO2 - concentrations but had no benefit on an intermittent running test that reflects the demands of rugby performance, and CMJ performances. The findings do not support acute high-dose NO3 - supplementation as an ergogenic aid to enhance physical performance in trained male rugby players. Highlights
 It has been suggested that NO3 - supplementation might have ergogenic potential for team sport athletes, however, the impact of nitrate supplementation on intermittent running performance of rugby players is unclear. Therefore, we aimed to assess the impact of acute NO3 - supplementation on the Prone Yo-Yo IR1 performance in trained male rugby players. Acute supplementation of high-dose nitrate supplementation substantially increased plasma NO3 - and NO2 - concentrations but did not improve the Prone Yo-Yo IR1 performance and countermovement jump performance in trained rugby players. The findings of the present study do not support acute BRJ supplementation as a nutritional ergogenic aid for trained rugby players, at least for the Prone Yo-Yo IR1performance, but are in line with the notion that supplementation of NO3 - is less likely to be ergogenic in well trained athletes.


Introduction
Dietary nitrate (NO 3 -) supplementation, most commonly in the form of beetroot juice, has become a popular ergogenic aid used by individual and team sport athletes for enhancing performance.Such supplementation increases circulating concentrations of NO 3 -and nitrite (NO 2 -), and subsequently nitric oxide (NO) bioavailability, which has a role in vasodilation (Jones et al., 2018).NO production via this NO 3 --NO 2 --NO pathway is accelerated in hypoxic and acidic environments such as that observed in skeletal muscle during exercise (Lundberg & Weitzberg, 2010).Indeed, improvements in submaximal exercise efficiency, high-intensity exercise tolerance, and fatigue resistance has been attributed to elevated plasma NO 2 -via supplementation of NO 3 - (Jones et al., 2018).NO 3 -supplemen-properties (Jones et al., 2018;Esen et al., 2022a), such as evoked contractile force and the rate of force development in type II muscle fibres (Hernández et al., 2012;Jones et al., 2018).Although it remains to be elucidated in humans, the improved muscle contractility has been attributed to increased perfusion and oxygenation (Ferguson et al., 2013), and improved calcium handling and release in type II fibres (Hernández et al., 2012).Taken together, it makes the expectation tenable that the ergogenicity of NO 3 -supplementation might be evident during intermittent exercise as it requires greater input of type II fibres (Krustrup et al., 2006(Krustrup et al., , 2009)).
The Yo-Yo intermittent recovery level 1 test (Yo-Yo IR1) is well-established, ecologically valid and commonly used (Krustrup et al., 2003) and heretofore, recent studies have shown an enhanced performance in the Yo-Yo IR1 test following varied supplementation regimen of NO 3 -(range of dose between 6.4 and 29 mmol; and range of duration between acute and 7days supplementation) in various populations (i.e.active adults, recreational or/and trained soccer, hockey players) (Wylie et al., 2013;Thompson et al., 2016;Nyakayiru et al., 2017;Esen et al., 2022b).Although these studies have concluded that NO 3 -supplementation might be an ergogenic aid for team sport athletes, translation of these findings to every team sport activity and/ or players is complicated due to the considerable differences in technical requirements and metabolic demand of each team sport.For example, rugby requires players to engage in high-impact collisions, wrestles and tackles in addition to intermittent running, leading to greater physical and physiological demands (Gabbett, 2005).Indeed, greater sub-maximal and peak metabolic, physiological and movement responses, but less covered total distance was reported during the Prone Yo-Yo IR1 test (starting each shuttle in the prone position) compared to standard Yo-Yo IR1 test in rugby players (Dobbin et al., 2018b).Given getting up from the floor at the start of each shuttle and accelerating would increase upper-and lower-body muscle activation, this additional movement could lead to a greater reliance on type II fibers, and subsequent metabolite accumulation that could accelerate fatigue (Dobbin et al., 2018a).Collectively, NO 3 -supplementation might be expected to enhance rugby performance as this physiological condition (greater type II fibers reliance and more acidic environment) during the prone Yo-Yo IR1 might facilitate the reduction of NO 2 -to NO (Modin et al., 2001) and thus augment ergogenic potential of NO 3 supplementation.However, to date, the effect of NO 3 supplementation on intermittent running performance in trained male rugby players is yet to be determined.This is important to evaluate because if similar findings after NO 3 -supplementation are evident during activity that reflects the demands of rugby, it could support a rugby-specific practical nutritional strategy for improving training quality and match performance.As rugby performance is multifaceted (Gabbett, 2005), intermittent running tests such as the Yo-Yo IR1 alone can only provide a limited replication and measurement of the physiological demands of the sport.The countermovement jump (CMJ) can be a practical and appropriate method commonly used to assess muscle power and fatigue (Twist & Highton, 2013).Within rugby it has also been observed to correlate with other outcomes such as tackle ability (Redman et al., 2022) and dynamic balance (Wilczyński et al., 2021).Several studies have investigated the effects of NO 3 -supplementation on CMJ in different recreational or/and athletic population (recreational or/and athletic) (Clifford et al., 2016(Clifford et al., , 2017;;Cuenca et al., 2018;Jonvik et al., 2021); however, the effect of NO 3 -supplementation on CMJ in rugby players remains to be determined.
Those recent studies reported conflicting findings regarding to the effect of NO 3 -on CMJ.Whilst some reported that NO 3 -attenuated the decrease in CMJ after a bout of repeated sprints (Clifford et al., 2016), some others reported no effect after a marathon (Clifford et al., 2017) or 30-sec Wingate test (Cuenca et al., 2018) or fatiguing isometric muscle contractions (Jonvik et al., 2021).It is difficult to draw conclusions regarding the efficacy of NO 3 -supplementation to enhance jumping performance because of inter-study differences in the exercise modality employed.As such, further research is required to assess the effect of NO 3 supplementation on CMJ in rugby players.
The aim of the present study was to determine the effect of acute NO 3 -supplementation, via beetroot juice, on the Prone Yo-Yo IR1 performance and CMJ in trained male rugby players.We hypothesized that acute high-dose NO 3 -supplementation would improve the total distance covered in the Prone Yo-Yo IR1 test as well as CMJ performance compared to placebo.

Participants
Twelve trained (McKay et al., 2022) male rugby union players (mean ± SD, age: 20 ± 4 years, body mass: 69.2 ± 6.3 kg, height: 175.4 ± 5.9 cm) participated in this study.Six out of 12 participants were members of senior academy team of the same rugby union club competing in England's top division League.They were playing in the Rugby Football Union Championship (2nd tier competition in England rugby).Other six out of 12 participants were playing in the level 3 league (National League 1) in England.All participants were also playing in British Universities & Colleges Sport Super Rugby league (the highest level of men's university rugby in UK).Participants had 9 ± 3 years of rugby experience and were involved in a regular training session with an average of 8 × 90-min sessions per week (5 × 90-min field-based and 3 × 90-min gymbased sessions), and a minimum of one competitive match per week during the investigation.Participants were non-smokers, free from injury and medical conditions to safely participate in the study.Participants were required to record their dietary intake 48 h prior to the first trial.A copy of this dietary record was given to the participants, and they were required to replicate their diet 48 h before the second trial.Participants were also asked to refrain the ingestion of any source of caffeine, alcohol, nutritional supplements, and any anti-inflammatory drugs (24 h), and from strenuous exercise (24 h) before each testing day.Participants were also required not to use antibacterial mouthwash throughout the testing period, as it eradicates commensal bacteria and therefore attenuates in the reduction of NO 3 -to nitrite in the oral cavity (Govoni et al., 2008).All participants were informed of the study procedures and provided written informed consent before participation in this study.This study was approved by the University Research Ethics Committee with all procedures conducted in accordance with the Declaration of Helsinki.

Experimental design
A randomised, counterbalanced, double-blind, placebocontrolled crossover design was used in the present study.Each participant completed two separate experimental trials (over an 8-days period) on an indoor fourth-generation artificial grass pitch under similar environmental conditions (temperature:18 ± 2 °C, humidity: 52.0% ± 2.3%, pressure: 1018 ± 2 mbar) at the similar time of the day (09:00-11:00).Before the experimental trials, the participants attended one familiarization session including all procedures (except blood sampling and supplement administration).The data during this familiarization session were not used for further analyses.Participants were instructed to arrive at the testing facility in a rested and fully hydrated state, ≥ 2 h post-prandial.All participants were wellexperienced with the prone Yo-Yo IR1 test.The Experimental trials were performed following acute supplementation of either concentrated NO 3 --rich (BRJ) or NO 3 --depleted (PLA) beetroot juice and interspaced by one week wash-out as suggested by Wylie et al. (2013b).Supplements were allocated in a randomised order and double-blind design by an independent technician who had no further involvement in this study.The randomization and blinding were held until the end of the study.Participants were also asked at the end of the study whether they could tell which trial they were on to check allocation concealment.Fifty eight percent said they did not know, 25% thought they were on BRJ, and 17% thought they consumed placebo.Of the participants who expressed an opinion, 60% were incorrect in their selection of supplement.Hence, the study blinding was effective.

Procedure
On the first experimental day, participants arrived at the testing facility 2.5 h after consuming the supplements.Upon arrival, 4 mL blood was collected from the cubital fossa vein into a lithium-heparin tube (Vacutainer, Becton Dickinson).In addition, a capillary blood lactate (BLa) sample was obtained before (pre-BLa) and immediately after (post-BLa) the performance testing from finger pinprick sample.Then, all participants performed a standardized warm-up for 10 min, ending with the first two shuttles of the prone Yo-Yo IR1 test, for familiarization to the audio and initial speeds, followed by a 5 min rest in a seated position.Performance testing consisted of CMJs without arm swing (pre-CMJ), the Prone Yo-Yo IR1 test, and another CMJs (post-CMJ).

The Prone Yo-Yo IR1 test
The Prone Yo-Yo IR1 test was performed as described elsewhere (Dobbin et al., 2018a).In brief, it consists of repeated shuttle (2 × 20 m) runs, separated by a 10 s active recovery period (5 m jog, 180°change in direction and walk to the line), at progressively increasing speeds controlled by audio bleeps from a portable audio system.Each running bout was started from a prone position in which participants' head were behind the start line, legs were straight, and chest were in contact with the ground.The speed of the shuttles was 10-13 km•h −1 at first four (0-160 m), at 13.5 km•h −1 next three (200-280 m), and at 14.0 km•h −1 at next four (320-440 m).After that, the speed increased 0.5 km•h −1 every eight shuttles (i.e.760, 1080, 1400 m, etc.).When participants failed twice to reach the start/finish line in the allocated, distance covered was recorded as the final distance.The coefficient of variation (CV) and intra-class correlation coefficient of the prone Yo-Yo IR1 has been reported as 9.9% and 0.98, respectively (Dobbin et al., 2018b).

CMJ
Participants performed three CMJ to assess jump height (cm) using optojump system (Optojump, Microgate, Bolzano, Italy), which calculates height (cm) via flight time.Participants were instructed to stand in upright position, squatted down to a 90°knee angle, then immediately jumped upwards as fast and high as they can.Three repetitions were separated by 30 sec standing rest.The highest jump heights (cm) recorded as result measures.Test-retest measurements revealed a CV of 2.1%.

Measurements
A venous blood samples were taken ∼3 h after ingestion of supplement and immediately centrifuged at 1160 g and 4°C for 10 min (ettich® 320 centrifuge, Canada).Plasma was then extracted and stored in labelled tubes at −80°C for later analysis of to assess plasma NO 3 -and NO 2 -concentrations using a modification of the chemiluminescence technique via chemiluminescence analyser (the Sievers NOA 280i, Analytix, UK) as described elsewhere (Shannon et al., 2016;Wang et al., 2022).Pre-and post-BLa sample was taken from the pad of the index finger of the left hand and analysed using the Lactate Pro-2 (Lactate Pro analyser, Arkay, Kyoto, Japan).

Statistical analysis
Sample size was calculated, by using G*Power software (version 3.1.9.4,Universität, Düsseldorf, Germany), based on research examining the effect of beetroot juice supplementation on Yo-Yo IR-1 performance in soccer players (Nyakayiru et al., 2017).T-test family was used with matched pairs, a two-sided significance level of 0.05, a power of 0.80 and a standardized mean difference of 1.03 indicated that 12 participants would be required.Normality of all data was confirmed via the Shapiro-Wilk test.The differences between the PLA and BRJ supplements in the Prone Yo-Yo IR1 test performance, and plasma NO 3 -and NO 2 -concentrations were determined by employing paired samples t-tests.The magnitude of difference between PLA and BRJ conditions was measured with Cohen's dz (Lakens, 2013) effect sizes and interpreted as follows: large d > 0.8, moderate d = 0.8-0.5, small d = 0.5-0.2, and trivial d < 0.2 (Cohen, 1988).A two-way (supplement × time) repeatedmeasures ANOVA was used to analyse CMJ and BLa before and after the intermittent exercise following PLA and BRJ supplementation.Statistical significance was set at p < 0.05, and all data were analysed using SPSS 28.0 (IBM Corp., Armonk, NY), and are presented as mean ± SD.
The effects of BRJ and PLA supplementation on CMJ and BLa are shown in Table 1.There was a main effect of time in CMJ (P < 0.001, dz = 0.56) showing that vertical jump height reduced after the prone Yo-Yo IR1 test.There was neither a main effect of supplement nor an interaction effect in CMJ at any point (all P > 0.05).There was a main effect of time in BLa (P < 0.001, dz = 5.5) showing that BLa increased after the prone Yo-Yo IR1 test.There was no either main effect of supplement or interaction effect in BLa at any point (all P > 0.05).

Discussion
This is the first study to investigate the effect of acute NO 3 -supplementation (12.8 mmol/d-1), in the form of BRJ, on an intermittent running performance test that reflects the demands of the sport (the Prone Yo-Yo IR1) in trained male rugby players.The main results showed that acute supplementation of BRJ increased plasma NO 3 -and NO 2 -concentration but had no effect on the Prone Yo-Yo IR1 test, CMJ or Bla.These results indicate that acute (high) dose of 12.8 mmol NO 3 -of BRJ supplementation does not result in a meaningful ergogenic benefit on trained male rugby players.Plasma NO 3 -and NO 2 -were elevated by 768% and 310%, respectively, following acute (3 h prior to exercise) supplementation of 12.8 mmol NO 3 -compared to PLA.
These findings are in agreement with previous research that observed increases to a similar extent in both plasma NO 3 -and NO 2 -following acute NO 3 -supplementation regimens (Wylie et al.,203b).Given the pharmacokinetic responses of plasma NO 3 -and NO 2 -following acute dietary NO 3 -supplementation (with similar doses that used in the present study) has been well reported (Wylie et al.,203b), the findings of the present study suggest an enhancement in NO bioavailability that was previously shown to improve performance.Acute supplementation of BRJ did not provide a significant effect on the prone Yo-Yo IR1 test performance in the present study.This finding is in contrast to recent reports that BRJ supplementation improved the total distance covered in the standard Yo-Yo IR1 test in recreational and trained participants (Wylie et al., 2013;Thompson et al., 2016;Nyakayiru et al., 2017;Esen et al., 2022b).Mostly based on those previous studies above, NO 3 -supplementation is suggested as an ergogenic aid for improving team sport performance.
However, since there are considerable differences among team sports regarding to required specific skills, and physiological demands of activities, it is not convincing to suggest potential ergogenicity of NO 3 -supplementation for every team sport.Therefore, for the first time, we applied a Yo-Yo test in trained rugby players to reflect rugby demands more closely.Therefore, our findings provide a significant contribution regarding to the ergogenic potential of NO 3 -supplementation over rugby-specific exercise in trained rugby male players and in line with previous studies in different team-sports athletes (i.e.basketball or hockey) that reported no ergogenic effect following acute BRJ supplementation (López-Samanes et al., 2020;2022a;2022b).Acute BRJ supplementation had no effect jump height during CMJ before the Prone Yo-Yo IR1 test in the present study.Consistent with our findings, no effect of BRJ supplementation was found on CMJ in a non-fatigued state (pre-exercise) in previous studies (Cuenca et al., 2018;Jonvik et al., 2021).The present study has been also unable to detect any effect of BRJ supplementation on jump height during CMJ after the Prone Yo-Yo IR1 test.This result is also in line with some, but not all (Clifford et al., 2016;López-Samanes et al., 2022b), studies that reported no effect of BRJ on the decrement in CMJ   The main of time (P < 0.05).
following fatiguing exercise (Clifford et al., 2017;Cuenca et al., 2018).It remains to be elucidated, but there could be several possible explanations for the absence of an effect of BRJ on performance in the present study.It has been a topic of debate whether benefits of NO 3 -supplementation would be dependent on fitness or/and training status of individuals (Senefeld et al., 2020;Shannon et al., 2022).Since there is evidence that has showed the ergogenic potential of NO 3 -is associated with the individual aerobic fitness level, with no benefits observed on trained subjects (Porcelli et al., 2015), this might be attributed to the lack of effect of NO 3 -in the present study.Most previous research above who reported ergogenic benefits of NO 3 -on the standard Yo-Yo IR1 or/and CMJ performances applied short-term BRJ supplementation periods (36 h -7-day) (Wylie et al., 2013;Thompson et al., 2016;Nyakayiru et al., 2017); compared to the present study.Thus, duration of supplementation may have caused the effect observed by those previous studies.However, the present study particularly aimed to evaluate the acute effect of NO 3 -supplementation, as it would be a more practical and applicable nutritional strategy for team sports athletes.Although some recent meta-analyses have highlighted that acute or short-term supplementation regimen do not differ regarding to performance enhancing potential of NO 3 - (Senefeld et al., 2020;Esen at al., 2022a), multiple-day supplementation is currently suggested for trained athletes (Shannon et al., 2022).Further, it is suggested that high-intensity intermittent exercise performance may be more likely to be improved with short term as opposed to acute nitrate supplementation (Alsharif et al., 2023).Therefore, it cannot be ruled out that multiple-day supplementation would have resulted in an improvement following BRJ.
The present study has several limitations that should be addressed.Although we aimed consciously not to interfere in dietary intake behaviour to reflect real-life practice, particularly as the amount of administered NO 3 -was much higher than its ingestion in the regular diet (∼ 0.7-5 mmol) (Gangolli et al., 1994;Mensinga et al., 2003;Hord et al., 2009), there could be an interaction between nutrient intake pre-trial and the influence of NO 3 -supplementation.However, the crossover design should negate some of this limitation.We were unable to control participants' dietary intake so instead instructed them to record their diet in the 48 h before the first trial to allow replication before the subsequent trial, which each participant confirmed.

Practical application
Our findings indicate that acute NO 3 -supplementation via BRJ does not provide an ergogenic benefit for trained rugby players, at least for intermittent running performance and CMJ, but are in line with the notion that supplementation of NO 3 -is less likely to be ergogenic in well trained athletes (Shannon et al., 2022).However, practitioners are encouraged to assess the performance effects of NO 3 -supplementation during longer durations, as this may affect the ergogenic potential of NO 3 -on rugby players.

Conclusion
Acute supplementation of NO 3 --rich BRJ (12.8 mmol NO 3 -) increased plasma NO 3 -and NO 2 -concentrations but had no benefit on intermittent running test that reflects the demands of rugby performance, and CMJ performances in trained male rugby players.The findings of the present research do not support acute high-dose NO 3 -supplementation as an ergogenic aid to enhance physical performance in trained male rugby players.

Figure 1 .
Figure 1.Plasma nitrate (NO 3 -, panel A) and nitrite (NO 2 -, panel B) concentration responses after acute beetroot juice (BRJ) or placebo (PLA) supplementation.Group means (SD) are shown in black lines and individual responses are shown in dashed lines.*P < 0.05.

Figure 2 .
Figure 2. Group mean (SD) and individual the prone Yo-Yo IR1 test performances (the total distance covered) after acute beetroot juice (BRJ) or placebo (PLA) supplementation are shown in the black and dashed lines, respectively.

Table 1 .
Effects of beetroot juice (BRJ) and placebo (PLA) supplementation on countermovement jump (CMJ) and blood lactate concentration (BLa) before and after the Prone Yo-Yo IR1 test.