Acute Beetroot Juice Supplementation Enhances Intermittent Running Performance but Does Not Reduce Oxygen Cost of Exercise among Recreational Adults

ate (NO 3 ) supplementation has been reported to enhance intermittent exercise perfor- mance; however, its impact on oxygen (O 2) cost during intermittent running exercise is unclear. The aim of this study was to assess if acute NO 3 supplementation would elicit performance bene ts in recreationally active individuals during the Yo–Yo intermittent recovery level 1 (Yo-Yo IR1) test, with its potential bene t on O 2consumption (VO 2), in a double-blind, randomized, crossover study, 12 recreational males consumed NO 3 -rich (NIT; ~12.8 mmol), and NO 3 -depleted (PLA; 0.04 mmol) concentrated beetroot juice 3 h before completing the Yo-Yo IR1 test. VO 2was measured at 160, 280 and 440 m (sub-maximal) and when the test was terminated (peak). Performance in the Yo–Yo IR1 was greater with NIT (990 442.25 m) compared to PLA (870 357.4 m,p= 0.007). The VO 2 was not signi cantly different at 160 m (1.92 0.99 vs. 2.1 0.88 L min 1 ), 280 m (2.62 0.94 vs.2.83 0.94 L min 1 ), 440 m (3.26

(sub-maximal) and when the test was terminated (peak). Performance in the Yo–Yo IR1 was greater with NIT (990 442.25 m) compared to PLA (870 357.4 m,p= 0.007). The VO 2 was not signi cantly different at 160 m (1.92 0.99 vs. 2.1 0.88 L min 1 ), 280 m (2.62 0.94 vs.2.83 0.94 L min 1 ), 440 m (3.26 1.04 vs. 3.46 0.98 L min 1 ) and peak (4.71 1.01 vs. 4.92 1.17 L min 1 ) between NIT and PLA trials (allp> 0.05). The present study has indicated that acute supplementation of NO 3 enhanced intermittent running performance but had no effect on VO 2during the Yo–Yo IR1 test in recreational young adults. Keywords:nitric oxide; ergogenic aid; sports nutrition; beetroot juice; exercise performance 1. Introduction The ergogenic effect of dietary nitrate (NO3 ) supplementation is attributed to its reduction of NO3 to nitrite (NO2 ) and, subsequently, nitric oxide (NO) [1]. This ingestion of NO3 -rich sources is known to increase plasma NO2 and be bene cial for reducing the oxygen (O2) cost for a given workload [2–5], improving muscle contractile properties [6,7], and supporting fatigue resistance [8–10]. Interestingly, it has been shown that a vegetable source is more effective than NO3 salts [11], taken as a supplement (e.g., concentrated beetroot juice). Furthermore, existing evidence also supports the notion that the reduction of NO2 to NO is enhanced in intra-muscular hypoxic conditions such as that observed within the skeletal muscle during high-intensity activity [12,13].The potential bene ts of NO3 supplementation were also shown in muscle contractile properties (e.g., evoked contractile force), and these effects seem in preferentially type II compared to type I muscle bers [14]. This enhancement in muscle contractility after NO3 supplementation has been attributed to improved calcium handling and release [6,7] and improved skeletal muscle blood ow and vascular conductance during submaximal efforts [15]. As such, enhanced intermittent running performance in moderately- [16,17] and well-trained individuals [18] following NO3 supplementation might be associated with the potential type II bers' Nutrients2022,14, 2839.

to improved calcium handling and release [6,7] and improved skeletal muscle blood ow and vascular conductance during submaximal efforts [15]. As such, enhanced intermittent running performance in moderately- [16,17] and well-trained individuals [18] following NO3 supplementation might be associated with the potential type II bers' Nutrients2022,14, 2839.

Nutrients2022,14, 2839 2 of 10 speci c impact of NO3 [6,15] as type II bers are predominantly recruited to satisfy the high muscle contraction demands during high-intensity intermittent exercise [19]. The Yo–Yo intermittent recovery level 1 test (Yo–Yo IR1) is well-established, ecolog- ically valid and commonly used [20]. There are three previous studies that investigated the in uence of NO3 on the Yo–Yo IR1 test. Wylie et al. [16] reported an improvement in the Yo–Yo IR1 by 4.2% amongst moderately-trained team sport players following very large dose of NO3 (29 mmol) over 36 h before the test. Subsequent studies observed similar enhancement (3.9% and 3.4%) in the Yo–Yo IR1 test after the ingestion of 6.4 mmol for 5 days in moderately-trained team sport players [17] and a 12.8 mmol/day for 6 days in highly trained soccer players, respectively [18]. Besides the heavy recruitment of type II bers during a transition from low to high metabolic rate [21,22], intermittent running also leads to a high O2demand relative to O2delivery. Therefore, combined with the possible effects on type II bers, the enhanced Yo–Yo IR1 performance in the previous studies might also link to the potential bene ts of NO3 supplementation on exercise ef ciency by reducing the O2cost [8]. Oxygen consumption (VO2) at a given velocity is an endurance performance referred as running economy [23], and it is assessed because it re ects the energy cost of running [24]. Whilst most studies have reported an enhanced economy using acute and/or multiple days of NO3 supplementation during steady-state endurance exercises [25], it is presently unclear whether the enhanced economy effect of NO3 supplementation that was observed during steady-state endurance exercises might also occur during an intermittent exercise as the Yo–Yo IR1 test. As such, further research is required to assess the effect of NO3 supplementation on the response of VO2during intermittent running in humans. The previous studies above which reported enhanced intermittent running perfor- mance applied short-term supplementation periods [16–18], but previous meta-analyses reported that there is no difference between acute (e.g., 2–3 h pre-exercise) and chronic dosing regimens (e.g., 1–15 days) of

such, further research is required to assess the effect of NO3 supplementation on the response of VO2during intermittent running in humans. The previous studies above which reported enhanced intermittent running perfor- mance applied short-term supplementation periods [16–18], but previous meta-analyses reported that there is no difference between acute (e.g., 2–3 h pre-exercise) and chronic dosing regimens (e.g., 1–15 days) of NO3 on endurance exercise [26,27]. In addition, more recent meta-analyses have revealed that the bene ts of NO3 supplementation in power output generally are apparent following acute supplementation [14,28]. Despite consistent effects of NO3 supplementation on the Yo–Yo IR1 test after multiple-day supplementation, to date, its effect after acute supplementation is yet to be determined. This is important to evaluate, as it would be a more practical and applicable nutritional intervention approach for performance in team sports. Therefore, the aim of this study was to assess if acute supplementation of NO3 would elicit performance benefits in recreationally active individuals during the Yo–Yo IR1 test, with its potential benefit on VO2consumption. It was hypothesized that NO3 supplementation would enhance performance and reduce VO2cost during the Yo–Yo IR1 test. 2. Materials and Methods 2.1. Participants The sample size of this study was based on a priori calculation using G*Power soft- ware (version 3.1.9.4, Universität, Düsseldorf, Germany). In determining the minimal estimated sample, we have considered two key outcomes, namely total distance achieved and the difference in overall change of VO2. Considering total distance, a standardized mean difference of 1.03 was used based on the work of Nyakayiru et al. [18]. Considering the difference in change of VO2, a standardized mean difference of 0.93 was used based on the work of Bailey et al. [8]. In both instances, at-test family was used with matched pairs, a power of 0.80, a two-tailed approach, and was set at 0.05. The results from these estima- tions indicated that sample of 10–12 participants would be suf cient to detect a difference between NO3 (NIT) and placebo (PLA) supplementation. Twelve recreationally active males (mean SD: age 27 10 years, body mass 78.1 11.8 kg, stature180.5 5.3 cm )

matched pairs, a power of 0.80, a two-tailed approach, and was set at 0.05. The results from these estima- tions indicated that sample of 10–12 participants would be suf cient to detect a difference between NO3 (NIT) and placebo (PLA) supplementation. Twelve recreationally active males (mean SD: age 27 10 years, body mass 78.1 11.8 kg, stature180.5 5.3 cm ) were recruited for this study. All participants were involved in regular moderate-intensity exercise ~3 days per week and muscle-strengthening activities ~2 days per week. The participants were non-smokers, healthy, and did not use dietary supplements at the time of

Nutrients2022,14, 2839 3 of 10 data collection. All participants were university students in the Sport Science department and were familiar with the Yo–Yo IR1 test. Ethics approval for this study was given by the Manchester Metropolitan University Research Ethics Committee (Reference no: 33132). All participants were informed of the nature and possible risks of the experimental procedures before providing written informed consent. 2.2. Experimental Design The participants visited the testing facility on two separate occasions. Participants were assigned in a randomized, double-blind, placebo-controlled, crossover design to consume either a NO3 -rich (NIT) or a NO3 -depleted concentrated beetroot juice (PLA). The experimental trials were all carried out at the similar time of the day ( 2 h). Mean and standard deviation of ambient temperature, humidity and pressure during the two trials were 17 2.1 C, 56.0 4.3% and 1018 2 mbar, respectively. A four- to six-day washout period separated the supplementation periods, as suggested by Wylie et al. [9]. Each participant was asked to record their dietary intake in the 24 h before the rst experimental trial and replicate this in the 24 h before the subsequent trial. Participants were instructed to avoid strenuous exercise and the consumption of alcohol and caffeine for at least 24 h before each experimental trial. Participants avoided using antibacterial mouthwash throughout the duration of the study due to its prevention of the reduction of NO3 to NO2 in the oral cavity [29]. 2.3. Supplementation Protocol The participants received 140 mL of concentrated beetroot juice (~12.8 mmol of NO3 ; Beet it, James White Drinks Ltd., Ipswich, UK) or NO3 -depleted beetroot juice (~0.04 mmolof NO3 ) as PLA. This chosen dose was based on current recommendation for the optimal ergogenic effect of NO3 supplementation (5–13 mmol of NO3 ) [1,9,27]. Participants consumed 2 70 mL the supplement 3 h before the test to coincide with peak plasma [NO2 ] [9]. 2.4. Procedures Upon arrival at the testing facility (an indoor fourth-generation arti cial grass pitch), participants completed a standardized warm-up (10 min), ending with the rst two shuttles of the Yo–Yo IR1 test in

(5–13 mmol of NO3 ) [1,9,27]. Participants consumed 2 70 mL the supplement 3 h before the test to coincide with peak plasma [NO2 ] [9]. 2.4. Procedures Upon arrival at the testing facility (an indoor fourth-generation arti cial grass pitch), participants completed a standardized warm-up (10 min), ending with the rst two shuttles of the Yo–Yo IR1 test in order to familiarize themselves with the audio and initial speeds. After 10 min of passive recovery, a resting capillary blood lactate (BLa) sample was taken from the pad of the index nger of the left hand using the Lactate Pro-2 (Lactate Pro analyser, Arkay, Kyoto, Japan). Immediately after the Yo-Yo IR1, a second capillary BLa sample was taken. Participants had an online gas analyzer tter using a custom-made harness with the device positioned on their back. Pulmonary gas exchange was measured continuously using a Cosmed K5 (Cosmed, K5, Cosmed, Rome, Italy) with the system set for breath- by-breath analysis. The calibration of the K5 gas analyzer was performed before each test, according to the manufacturer's instructions including a gas, volume, carbon dioxide (CO2) and breathing frequency. Breath-by-breath VO2, CO2production (VCO2) and minute ventilation (VE) data from each test were linearly interpolated to provide second-by-second values. Subsequently, mean VO2, VCO2and VE were assessed during each run and recovery period and averaged to provide the overall mean VO2, VCO2and VE during the run and recovery periods for each stage of the Yo–Yo IR1 test. The mean sub-maximal values of 160, 280 and 440 m were based on those previously used [30]. Peak values for each variable were considered as the highest value achieved during the test. Previous literature has reported that the COSMED K5 had excellent reliability for VO2(CV: 4.4%, CI: 3.2–6.7%, concordance correlation coef cient [CCC]: 0.95), VCO2(CV: 6.2%, CI: 4.5–9.7%, CCC: 0.92) and VE (CV: 6.9%, CI: 4.9–10.7%, CCC: 0.89) during more than 2 h of continuous eld tests [31].

concordance correlation coef cient [CCC]: 0.95), VCO2(CV: 6.2%, CI: 4.5–9.7%, CCC: 0.92) and VE (CV: 6.9%, CI: 4.9–10.7%, CCC: 0.89) during more than 2 h of continuous eld tests [31].

Nutrients2022,14, 2839 4 of 10 The Yo–Yo IR1 test has been described elsewhere [20]. Brie y, the test consists of repeated 2 20 m runs, interspersed by a 10 s active recovery period, at progressively increasing speeds controlled by audio bleeps from a portable audio system. First four shuttles were at the speed of 10–13 km h 1 (0–160 m), then three shuttles at 13.5 km h 1 (200–280 m) and four shuttles at 14.0 km h 1 (320–440 m); thereafter, the speed increased 0.5 km h 1 every eight shuttles (i.e., 760, 1080, 1400 m, etc.). The nal distance successfully covered was recorded after the second failed attempt to meet the start/ nish line in the allocated time. 2.5. Statistical Analysis All data were presented as means SD. Differences between NIT and PLA in distance covered during the Yo–Yo IR1 test, VO 2peak, VCO 2peakand VE peak, RER peakand BLa pre- and post-exercise test were analyzed using a paired samplest-test. Effect sizes (d) were calculated through Cohen'sdas: larged> 0.8, moderated= 0.8 to 0.5, smalld= 0.5 to 0.2, and triviald< 0.2 [32]. Differences in VO2, VCO2, VE and RER at 160, 280 and 440 Im were determined using two-way repeated-measure ANOVA (supplement distance). In addition, effect size was calculated as partial eta-squared (Nutrients 2022, 14, x FOR PEER REVIEW 4 of 11 CCC: 0.92) and VE (CV: 6.9%, CI: 4.9–10.7%, CCC: 0.89) during more than 2 h of continu- ous field tests [31]. The Yo–Yo IR1 test has been described elsewhere [20]. Briefly, the test consists of repeated 2 × 20 m runs, interspersed by a 10 s active recovery period, at progressively increasing speeds controlled by audio bleeps from a portable audio system. First four shuttles were at the speed of 10–13 km·h −1 (0–160 m), then three shuttles at 13.5 km·h −1 (200–280 m) and four shuttles at 14.0 km·h −1 (320–440 m); thereafter, the speed increased 0.5 km·h −1 every eight shuttles (i.e., 760, 1080, 1400 m, etc.). The final distance successfully covered was recorded after the second failed attempt to meet the start/finish line in the

speed of 10–13 km·h −1 (0–160 m), then three shuttles at 13.5 km·h −1 (200–280 m) and four shuttles at 14.0 km·h −1 (320–440 m); thereafter, the speed increased 0.5 km·h −1 every eight shuttles (i.e., 760, 1080, 1400 m, etc.). The final distance successfully covered was recorded after the second failed attempt to meet the start/finish line in the allocated time. 2.5. Statistical Analysis All data were presented as means ± SD. Differences between NIT and PLA in distance covered during the Yo–Yo IR1 test, VO 2peak, VCO2peak and VEpeak, RERpeak and BLa pre- and post-exercise test were analyzed using a paired samples t-test. Effect sizes (d) were calcu- lated through Cohen’s d as: large d > 0.8, moderate d = 0.8 to 0.5, small d = 0.5 to 0.2, and trivial d < 0.2 [32]. Differences in VO 2, VCO2, VE and RER at 160, 280 and 440 Im were determined using two-way repeated-measure ANOVA (supplement × distance). In addi- tion, effect size was calculated as partial eta-squared (ŋ p 2) varying small (<0.25), medium (0.26–0.63) and large (>0.63) [33]. All data were analyzed using SPSS 27.0 (IBM Corp., Ar- monk, NY, USA). Significance was determined at p < 0.05. 3. Results The distance covered in the Yo–Yo IR1 test was significantly greater in NIT (990 ± 442.25 m) compared to PLA (870 ± 357.4 m, p = 0.007, d = 0.30, Figure 1). Figure 1. The distance covered in the Yo–Yo IR1 test was 14% greater with NIT compared to PLA. The dashed lines indicate the responses of individual participants. The solid line indicates the group mean (±SD). * p < 0.05. The group mean relative and absolute VO2 and absolute VCO2 responses at 160m, 280m, 440m and peak during the Yo–Yo IR1 following both NIT and PLA supplementa- tion were shown in Figure 2, and values (including VE and RER values) were reported in Table 1. The mean relative VO 2 responses at submaximal distances were similar for NIT p 2 ) varying small (<0.25), medium (0.26–0.63) and large (>0.63) [33]. All data were analyzed using

and peak during the Yo–Yo IR1 following both NIT and PLA supplementa- tion were shown in Figure 2, and values (including VE and RER values) were reported in Table 1. The mean relative VO 2 responses at submaximal distances were similar for NIT p 2 ) varying small (<0.25), medium (0.26–0.63) and large (>0.63) [33]. All data were analyzed using SPSS 27.0 (IBM Corp., Armonk, NY, USA). Signi cance was determined atp< 0.05. 3. Results The distance covered in the Yo–Yo IR1 test was significantly greater in NIT (990 442.25 m ) compared to PLA (870 357.4 m,p= 0.007,d= 0.30, FigureNutrients 2022, 14, x FOR PEER REVIEW 4 of 11 CCC: 0.92) and VE (CV: 6.9%, CI: 4.9–10.7%, CCC: 0.89) during more than 2 h of continu- ous field tests [31]. The Yo–Yo IR1 test has been described elsewhere [20]. Briefly, the test consists of repeated 2 × 20 m runs, interspersed by a 10 s active recovery period, at progressively increasing speeds controlled by audio bleeps from a portable audio system. First four shuttles were at the speed of 10–13 km·h −1 (0–160 m), then three shuttles at 13.5 km·h −1 (200–280 m) and four shuttles at 14.0 km·h −1 (320–440 m); thereafter, the speed increased 0.5 km·h −1 every eight shuttles (i.e., 760, 1080, 1400 m, etc.). The final distance successfully covered was recorded after the second failed attempt to meet the start/finish line in the allocated time. 2.5. Statistical Analysis All data were presented as means ± SD. Differences between NIT and PLA in distance covered during the Yo–Yo IR1 test, VO 2peak, VCO2peak and VEpeak, RERpeak and BLa pre- and post-exercise test were analyzed using a paired samples t-test. Effect sizes (d) were calcu- lated through Cohen’s d as: large d > 0.8, moderate d = 0.8 to 0.5, small d = 0.5 to 0.2, and trivial d < 0.2 [32]. Differences in VO 2, VCO2, VE and RER at 160, 280 and 440 Im were determined using two-way repeated-measure ANOVA (supplement × distance). In addi- tion, effect size was calculated as partial eta-squared (ŋ p

This study assesses the effects of acute NO3− supplementation on performance in recreationally active individuals.