Influence of Sex and Acute Beetroot Juice Supplementation on 2 KM Running Performance

e: To assess the effect of acute nitrate-rich (BJ) and nitrate-depleted (PL) beetroot juice ingestion on 2 km running performance in amateur runners, and to what extent the ergogenic effect of BJ supplementation would be in uenced by the sex of the participants; Methods: Twenty- four amateur long-distance runners (14 males and 10 females) performed a 2 km time trial (TT) on an outdoor athletics track 2.5 h after ingesting either 140 mL of BJ (~12.8 mmol NO 3 ) or PL. After the tests, blood [lactate] and ratings of perceived exertion (RPE) related to the leg muscles (RPE muscular), cardiovascular system (RPE cardio) and general overall RPE (RPE general) were assessed; Results: Compared to PL, BJ supplementation improved 2 km TT performance in both males (p< 0.05) with no supplement sex interaction effect (p> 0.05). This improvement in 2 km running performance was

tests, blood [lactate] and ratings of perceived exertion (RPE) related to the leg muscles (RPE muscular), cardiovascular system (RPE cardio) and general overall RPE (RPE general) were assessed; Results: Compared to PL, BJ supplementation improved 2 km TT performance in both males (p< 0.05) with no supplement sex interaction effect (p> 0.05). This improvement in 2 km running performance was a function of improved performance in the second 1 km split time in both males and females (p< 0.05). Supplementation with BJ did not alter post-exercise blood [lactate] (p> 0.05) but lowered RPE general(p< 0.05); Conclusions: acute BJ supplementation improves 2 km running performance in amateur runners by enhancing performance over the second half of the TT and lowering RPE generalby a comparable magnitude in males and females. Keywords: endurance performance; ergogenic aid; nitrate; nitric oxide; ratings of perceived exertion 1. Introduction Consumption of dietary supplements is a common pre-competition strategy for ath- letes competing in a variety of sports at different competitive standards in an attempt to enhance performance [1–6]. Although not unequivocal, there is evidence to suggest that dietary supplementation with inorganic nitrate (NO3 ) can enhance exercise performance in a variety of settings [7,8]. After ingestion, NO3 undergoes a stepwise reduction to nitrite (NO2 ) and then nitric oxide (NO), with the latter step augmented in conditions of acidosis and hypoxia, and believed to be responsible for the ergogenic effects of NO3 supplementation [7,9]. NO is a potent signalling molecule that elicits biological effects on numerous tissues and is involved in an array of physiological processes including, but not limited to, vasodilation, calcium handling, mitochondrial respiration, and neurotransmis- sion [9]. Most of the research assessing the ergogenic potential of NO3 supplementation to date has been conducted in males completing cycling exercise [10–27]. This experimental approach has limited wider understanding of the settings in which NO3 supplementation is more and less likely to be ergogenic. In studies assessing the effect of NO3 supplementation on running exercise responses, improved economy has been reported in recreationally active and moderately trained sub- jects, but not highly trained subjects [28–33], whereas improved

males completing cycling exercise [10–27]. This experimental approach has limited wider understanding of the settings in which NO3 supplementation is more and less likely to be ergogenic. In studies assessing the effect of NO3 supplementation on running exercise responses, improved economy has been reported in recreationally active and moderately trained sub- jects, but not highly trained subjects [28–33], whereas improved time to exhaustion has been Appl. Sci.2021,11, 977.

Appl. Sci.2021,11, 977 2 of 10 reported in both recreationally active [29] and highly trained [28] subjects. During running time trial (TT) performance tests in well trained endurance athletes, most studies [30,32–35] have observed no change in performance over distances spanning1.5–10 kmafter NO3 supplementation, but improved performance appears more likely over a shorter distance (1.5 km) compared to a longer distance (10 km) in this population after acute NO3 sup- plementation [36]. The effect of NO3 supplementation on running TT performance is less clear in recreationally active and moderately trained subjects with improvements in 3 km [32] and 5 km [37] performance having been observed in some studies, and no improvement in 5 km [36] and 10 km [38] performance having been observed in other studies. Therefore, further research is required to address the effect of NO3 supplementa- tion on middle distance (<5 km) running TT performance in moderately trained subjects. Moreover, since NO3 supplementation has been reported to improve performance over the rst half, but not the second half, of a 10 km TT [38], and to improve 5 km performance by mostly enhancing performance in the latter stages on the TT [36], further research is required to address which phases of TT performance are most likely to be enhanced by NO3 supplementation. To date, the majority of studies assessing the effect of NO3 supplementation on running performance have either used exclusively male subjects [32–34,38], or not reported data for each sex to allow potential sex-speci c effects of NO3 supplementation on running TT performance to be understood [30,36,37]. There is some evidence that, relative to males, females exhibit a greater increase in plasma [NO2 ] [39] and greater improvements in muscle contractile function [40] after NO3 supplementation. However, females present with a more oxidative skeletal muscle phenotype compared to males [41], which could blunt NO2 reduction to NO and the improvement in performance after NO3 supplementation. Although de Castro et al. [42] reported no effect of acute NO3 supplementation on 3 km running TT performance in untrained women, the NO3 supplement was ingested 30 min prior to exercise

females present with a more oxidative skeletal muscle phenotype compared to males [41], which could blunt NO2 reduction to NO and the improvement in performance after NO3 supplementation. Although de Castro et al. [42] reported no effect of acute NO3 supplementation on 3 km running TT performance in untrained women, the NO3 supplement was ingested 30 min prior to exercise such that plasma [NO2 ] would not have peaked until after the TT had been completed [43]. Therefore, further research is required to assess the potential for sex-speci c effects on the ef cacy of NO3 supplementation to improve running TT performance. The purpose of this study was to assess the effect of acute NO3 supplementation on 2 km running performance in recreational male and female runners. A 2 km running TT was selected as it is highly correlated with maximal aerobic speed [44], a key determinant of endurance performance [45]. It was hypothesized that, since plasma [NO2 ] increases to a greater extent in females after NO3 supplementation [39] and the increase in [NO2 ] post NO3 supplementation is correlated to improved performance [43], females would exhibit greater improvements in 2 km running performance compared to males. 2. Materials and Methods 2.1. Participants Twenty-four long-distance runners (14 males and 10 females) participated in the present study. All participants had been training in an amateur running club for at least two years prior to participating in this study. Participants' best times for the 10 km during the two months prior to data collection were 2382 338 s and 3127 359 s for male and female runners, respectively. The inclusion criteria for participation in this study were:(a) > 18 years; (b) free from any cardiovascular, muscular and/or bone pathology; (c) participating regularly in amateur running competitions of 10 km (at least one competition every 3 months); (d) having not taken any medicine or nutritional supplement within 3 months prior to the beginning of the study; (e) best personal time in10-km >30 min in males and 33 min in women. Prior to con rming their participation, all participants were informed of the

regularly in amateur running competitions of 10 km (at least one competition every 3 months); (d) having not taken any medicine or nutritional supplement within 3 months prior to the beginning of the study; (e) best personal time in10-km >30 min in males and 33 min in women. Prior to con rming their participation, all participants were informed of the study objectives and requirements. Two researchers answered any questions they had regarding their participation in the study before participants provided their written informed consent to participate in the study. Furthermore, participants were familiarised with the different perception scales which would be used subsequently

Appl. Sci.2021,11, 977 3 of 10 during the course of the study. This study was approved by a local ethics committee (Cod. 0045230632017) in agreement with the Declaration of Helsinki. 2.2. Anthropometric Measures A week prior to the start of performance testing, participants reported to the laboratory where an investigator completed an anthropometric assessment. The anthropometric measurements included stature, body mass, skinfold thickness (subscapular, biceps, triceps, suprailiac, supraspinale, front thigh, medial calf and abdominal), girths (arm, exed and tense, mid-thigh and calf) and breadths (humerus, bi-styloid and biepicondylar femur), which were conducted according to the recommendations of the International Society for the Advancement of Kinanthropometry (ISAK) with the laboratory temperature held constant at 24 C during these measurements. The formulas used to calculate body fat mass, muscle mass and bond mass were selected according to the recommendations for this population by the Spanish Kineanthropometry Group (GREC) [46]. 2.3. Design A randomized, double blind, crossover experimental design was employed to as- sess the effects of ingesting nitrate-rich (BJ) and nitrate-depleted (PL) beetroot juice on performance, blood lactate concentration ([BLa]) and ratings of perceived exertion (RPE) during 2 km running TT tests. Considering the differences in physiological response [47] and performance [48] according to the diurnal variations during running test, the two experimental sessions were conducted at the same time of day ( 0.5 h) and separated by 7 days, a suf cient period of time for ensuring a washout [49]. Weather conditions were similar for both testing sessions (sunny, wind speed average 1.18 0.9 m/s, 27 0.7 C and 25 4.2% humidity) and the tests were completed 667 m above sea level. 2.4. Warm-Up and 2 km Running Performance Tests Participants arrived at an outdoor 400 m synthetic athletics track 2.5 h before initiating the 2 km TT. A 2 km test was selected since this distance has been reported to be completed at a similar speed to the maximal aerobic speed [44] and it was a distance that participants were familiar with completing during their group interval training sessions. After ingesting either BJ or PL, participants completed a standardized 15

h before initiating the 2 km TT. A 2 km test was selected since this distance has been reported to be completed at a similar speed to the maximal aerobic speed [44] and it was a distance that participants were familiar with completing during their group interval training sessions. After ingesting either BJ or PL, participants completed a standardized 15 min warm-up of low-intensity running. Speci cally, they were instructed to run within an RPR range of 8 to 11 according to the 15-point (6–20) Borg scale [50], and subsequently completed 10 min of dynamic stretching and drills that was part of their usual warm up routine prior to training. The 2 km performance test was completed individually by participants and was hand timed to within 0.1 s by the coach and a researcher, with the mean time calculated and used for subsequent analysis. A 1 km split time was also obtained. Immediately after the 2 km TT, participants reported their RPE and a capillary blood sample was obtained within three min for [BLa] analysis. 2.5. Betroot Juice Supplementation and Diet Control Forty-eight hours prior to both 2 km TTs, participants were instructed to follow a standardized diet to ensure similar macronutrient (10% protein, 60% carbohydrate and 30% lipid) and micronutrient intake, in order to avoid dietary variation confounding our results. Furthermore, during the 24 h preceding both tests, participants were instructed to avoid the intake of NO3 -rich foods (beetroot, ruccula, celery, turnip, spinach, lettuce, leak, cabbage, parsley, endives) and caffeine. Participants were also instructed to avoid brushing their teeth and use of antibacterial mouthwashes for 24 h and 7 days, respectively, prior to both performance tests. On the experimental testing days, participants consumed 140 mL of a beetroot juice supplement that was either enriched in NO3 (~12.8 mmol NO3 ; BJ) (Beet IT; James White Drinks Ltd., Ipswich, UK) or a depleted of NO3 (PL), with the latter prepared as described previously [51]. The timing of BJ ingestion relative to the performance test was selected based on the pharmacokinetic data presented by

juice supplement that was either enriched in NO3 (~12.8 mmol NO3 ; BJ) (Beet IT; James White Drinks Ltd., Ipswich, UK) or a depleted of NO3 (PL), with the latter prepared as described previously [51]. The timing of BJ ingestion relative to the performance test was selected based on the pharmacokinetic data presented by

Appl. Sci.2021,11, 977 4 of 10 Wylie et al. [43] where plasma [nitrite] peaks 2–4 h post the ingestion of 140 mL of BJ. Participants started the standardized warm-up 135 min after supplement ingestion, and the 2 km time trial (TT) 150 min post supplementation ingestion, ensuring that the TT coincided with the plasma [nitrite] peak after supplementation. 2.6. Ratings of Perceived Exertion (RPE) The 15-point (6–20) Borg scale was used to record RPE [50]. The scale was “anchored” by explaining that a score of 20 should equate to a previous memory of absolute exhaus- tion. As described previously [52], participants were asked to indicate the RPE related to their leg muscles (RPE muscular), cardiovascular system (RPE cardio) and general overall RPE (RPE general). 2.7. Measurement of Blood Lactate Concentrations Post-test [BLa] was determined from capillarised blood samples (5 L) taken from the left index nger using the Lactate ProTM 2 LT-1710 blood analyzer (Arkray Factory Inc., KDK Corporation, Shiga, Japan). 2.8. Statistical Analysis Statistical analyses were performed using the Statistical Package for the Social Sciences 24.0 (IBM, Armonk, NY, USA). Data were initially checked for normality of distribution by Shapiro–Wilk test, equality of variances and sphericity as appropriate. When sphericity was violated, a Greenhouse–Geisser correction was employed. The 2 km completion times, 1 km split times, RPE and [BLa] were compared across supplementation conditions and sex using mixed model ANOVAs, with repeated measures for supplement. Effect sizes were calculated using partial eta-squared (h 2 p) considering values <0.25, 0.26–0.63 and >0.63 as small, medium and large effect sizes, respectively [53]. When a signi cant effect was detected, pairwise comparisons were assessed using the Holm–Bonferroni test. Pearson's correlation coef cient was used to determine whether 2 km completion time in the PL condition and the change in 2 km time between the PL and BJ conditions were related to determine whether the ergogenic potential of BJ supplementation was in uenced by baseline tness. Values are provided as mean (M) standard deviation (SD). Statistical signi cance was set atp< 0.05. 3. Results 3.1. Anthropometrics Measures The anthropometric data of the male

the PL condition and the change in 2 km time between the PL and BJ conditions were related to determine whether the ergogenic potential of BJ supplementation was in uenced by baseline tness. Values are provided as mean (M) standard deviation (SD). Statistical signi cance was set atp< 0.05. 3. Results 3.1. Anthropometrics Measures The anthropometric data of the male and female participants are presented in the Table. Table 1.Anthropometric data of the subjects. Variable Males Females Age (years) 38.7 9.2 36.6 8.2 Height (m) 1.74 0.05 1.64 0.08 Body mass (kg) 69.1 7.1 57.1 7.8 Body mass index (kg m 2 ) 22.8 2.2 21.2 2.2 Fat mass (kg) 8.9 2.9 11.0 3.6 Fat mass (%) 12.8 3.1 18.9 4.2 Muscle mass (kg) 36.1 2.6 25.8 2.5 Muscle mass (%) 52.6 4.1 45.5 3.3 Bone mass (kg) 12.4 2.3 9.4 0.9 Bone mass (%) 16.3 2.2 16.7 1.6 Data are shown as mean standard deviation. 3.2. Performance and Blood Lactate Concentrations There was a main effect for supplement on 2 km TT completion time and the second 1 km split time (p= 0.002,h 2 p= 0.372 and 0.282, respectively) with these values being

Appl. Sci.2021,11, 977 5 of 10 faster in the BJ condition compared to the PL condition (Table, Figure). There was a main effect of sex on 2 km TT performance and the rst and the second 1 km split times with these being faster in males than females (p< 0.001,h 2 p= 0.564–0.628) (Table). No differences across supplementation conditions and sex were found for [BLa] (Table). Table 2. Effect of nitrate-rich and nitrate-depleted beetroot juice supplementation on 2 km running performance and post-exercise blood lactate concentration. Parameter Condition Males Females Analysis Type F h 2 p 0–1 km (s) PL 213.6 26.6 276.1 32.4 Suppl. sex 0 0.014 BJ 214.4 26.1 276.9 30.5 Suppl. 0.322 0.014 Sex 28.427 0.564 § 1–2 km (s) PL 223.0 27.8 304.5 37.1 Suppl. sex 0.199 0.009 BJ 218.4 27.3 298.2 39.2 Suppl. 8.656 0.282 † Sex 37.1 0.628 § 2 km (s) PL 436.6 52.7 580.7 67.0 Suppl. sex 0.4 0.018 BJ 432.7 52.9 575.1 68.6 Suppl. 13.014 0.372 † Sex 34.023 0.607 § [BLa] (mmol/L) PL 15.2 5.0 15.6 2.7 Suppl. sex 0.011 0 BJ 15.8 6.1 16.4 3.7 Suppl. 0.489 0.022 Sex 0.077 0.003 Data are shown as mean standard deviation. Abbreviations: F: adjusted values for test statistic;h 2 p: partial eta squared; PL: nitrate- depleted juice; BJ: nitrate-rich beetroot juice; [BLa]: blood lactate concentration. Degrees of freedom were always 1 and 22. Signi cant differences were set atp< 0.05 ( † p< 0.01, § p< 0.001).Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 10 Bone mass (%) 16.3 ± 2.2 16.7 ± 1.6 Data are shown as mean ± standard deviation. 3.2. Performance and Blood Lactate Concentrations There was a main effect for supplement on 2 km TT completion time and the second 1 km split time (p = 0.002, ß ã 6 = 0.372 and 0.282, respectively) with these values being faster in the BJ condition compared to the PL condition (Table 2, Figure 1). There was a main effect of sex on 2 km TT performance and the first and the second 1 km split times with

Acute beetroot juice supplementation improves 2 km running performance in male and female amateur runners.