Effects of Beetroot Juice Supplementation on Cognitive Function, Aerobic and Anaerobic Performances of Trained Male Taekwondo Athletes: A Pilot Study

have shown that nitrate (NO 3 )-rich beetroot juice (BJ) supplementation improves endurance and high-intensity intermittent exercise. The dose–response effects on taekwondo follow- ing BJ supplementation are yet to be determined. This study aimed to investigate two acute doses of 400 mg of NO 3 (BJ-400) and 800 mg of NO 3 (BJ-800) on taekwondo-speci c performance and cognitive function tests compared with a placebo (PL) and control (CON) conditions. Eight trained male taekwondo athletes (age: 20 4 years, height: 180 2 cm, body mass: 64.8 4.0 kg) completed four experimental trials using a randomized, double-blind placebo-controlled design: BJ-400, BJ-800, PL, and CON. Participants consumed two doses of BJ-400 and BJ-800 or nitrate-depleted PL at2.5 h prior to performing the Multiple Frequency Speed of Kick Test (FSKT). Countermovement jump (CMJ) was performed before the (FSKT) and PSTT, whereas cognitive function was assessed (via the Stroop test) before and after supplementation and 10 min following PSTT. Blood lactate was collected before the CMJ tests immediately and 3 min after the FSKT and PSST; rating of perceived ex- ertion (RPE) was recorded during and after both speci c taekwondo tests. No signi cant differences (p> 0.05), with moderate and large effect sizes, between conditions were observed for PSTT and FSKT performances. In addition, blood lactate, RPE, heart rate, and CMJ height were not signi cantly different among conditions (p> 0.05). However, after the PSTT test, cognitive function was higher in BJ-400 compared to other treatments (p< 0.05). It was concluded that acute intake of 400 and 800 mg of NO

effect sizes, between conditions were observed for PSTT and FSKT performances. In addition, blood lactate, RPE, heart rate, and CMJ height were not signi cantly different among conditions (p> 0.05). However, after the PSTT test, cognitive function was higher in BJ-400 compared to other treatments (p< 0.05). It was concluded that acute intake of 400 and 800 mg of NO 3 rich BJ reported a moderate to large effect size in anaerobic and aerobic; however, no statistical differences were found in taekwondo-speci c performance. Int. J. Environ. Res. Public Health2021,18, 10202.

Int. J. Environ. Res. Public Health2021,18, 10202 2 of 17 Keywords:combat sport; ergogenic aid; high-intensity intermittent performance; martial arts 1. Introduction Taekwondo is an Olympic combat sport, with matches contested over three 2 min rounds separated by 1 min rest. It is a striking sport characterized by repeated high- intensity bursts of punching and predominantly kicking techniques lasting ~1–5 s, in- terspersed by low-intensity bouncing actions lasting ~10–35 s in a range from 1:2–1:7 work-to-rest ratios within each of the rounds [1]. Campos et al. [2] found that adenosine triphosphate (ATP) resynthesis during taekwondo competition is predominantly produced via the oxidative energy system (accounting for ~66 6% of total energy cost). However, both anaerobic glycolytic and ATP-PCr systems contribute to energy cost for ~4 2% and ~30 6% of total energy expenditure, respectively. Oxidative metabolism is the primary metabolic pathway during taekwondo combats, but the contribution of non-oxidative metabolism is likely to be the main source supporting repeated high-intensity kicking actions [3], which are required to score points and win matches. Nutritional supplement strategies that may improve performance or recovery as well as aiding oxidative and/or non-oxidative metabolism relevant in combat sport are of interest to taekwondo athletes [4]. However, studies have investigated dietary supplements, such as vitamins [5–7], carbo- hydrate drinks [8], caffeine [9], creatine monohydrate [10] and sodium bicarbonate [3,10] in the context of taekwondo, but no studies have analyzed the possible ergogenic effect of nitrate (NO3 )-rich beetroot juice (BJ) supplementation. BJ is an ergogenic aid that has received increased attention in the last decade, garnering strong support for enhancing exercise performance [11]. NO3 is a nitric oxide (NO) precursor and part of the NO3 — nitrite (NO2 )NO pathway [12], shown to modulate various aspects of physiological mechanisms, including muscle contraction and mechanical ef ciency [13]. Improvements in exercise capacity are suggested to occur due to a lowering of the cost of ATP to exercising muscles [14,15], increased vasodilation [16], and mitochondrial respiration, as well as intra- cellular calcium handling [17]. In addition, BJ diminishes the reduction of phosphocreatine (PCr) for a determined work [18], a

physiological mechanisms, including muscle contraction and mechanical ef ciency [13]. Improvements in exercise capacity are suggested to occur due to a lowering of the cost of ATP to exercising muscles [14,15], increased vasodilation [16], and mitochondrial respiration, as well as intra- cellular calcium handling [17]. In addition, BJ diminishes the reduction of phosphocreatine (PCr) for a determined work [18], a mechanism that may delay the depletion of PCr and favor the restoration of PCr during high-intensity intermittent efforts [19]. In addition, BJ supplementation may exert a central effect based on an increased level of tension and subjective vitality at the start of exercise [19,20] and improved physical performance with the same [21,22] or a lower [20,23] rating of perceived exertion (RPE). It is mediated by a possible increase in brain perfusion or a diminution of metabolite accumulation that atten- uate activation of III/IV muscle afferent feedback to the central nervous system and the potential for central fatigue development [23]. A meta-analysis found that dietary NO3 supplementation can improve endurance exercise performance [24] and improve cognitive function during exercises demanding quick and accurate decisions [25,26]. Additionally, increased power and force production in fast-twitch bers [17,27] and improved reaction time [27,28] were reported with intake of BJ. As with all supplements, determining the ideal dose and duration of the intake protocol is necessary to optimize ergogenic bene ts [29]. The International Olympic Committee (IOC) consensus statement suggests that an effective dosing protocol involves the acute ingestion of BJ in a range from 5 to 9 mmol (310–560 mg) dietary NO3 , 2–3 h pre-exercise [11], with lower dosages generally not showing bene cial effects on performance [30,31]. Taekwondo (and other combat sport) athletes may bene t from acute dietary NO3 ingestion due to the intermittent nature of combat sports [19], and the physical tness variables (such as strength, aerobic and anaerobic performance), and the cognitive functions required for successful performance in competition [1,32] Although positive effects of NO3 supplementation on handgrip strength in jiu-jitsu athletes [33] have been reported, the ergogenic effects of dietary NO3 ingestion on taekwondo or other striking combat

to the intermittent nature of combat sports [19], and the physical tness variables (such as strength, aerobic and anaerobic performance), and the cognitive functions required for successful performance in competition [1,32] Although positive effects of NO3 supplementation on handgrip strength in jiu-jitsu athletes [33] have been reported, the ergogenic effects of dietary NO3 ingestion on taekwondo or other striking combat sports performance are yet to be explored. In addition, the study that examined NO3

Int. J. Environ. Res. Public Health2021,18, 10202 3 of 17 supplementation on combat sports athletes utilized doses (12 mmol) well above that known to be generally ef cacious in other contexts [33]. Accordingly, the present study aimed to examine the effect of two acute doses of NO3 rich BJ (400 mg NO3 vs. 800 mg NO3 ) on aerobic and anaerobic taekwondo- speci c performance, RPE, blood lactate, and cognitive function. The hypothesis was that the ingestion of a high dosage (800 mg NO3 ) would increase taekwondo performance compared to a low dosage (400 mg NO3 ) during speci c taekwondo tests. However, high and low dosage of BJ supplementation would increase both cognitive function and exercise performance compared to placebo (PL) and control (CO) conditions. 2. Materials and Methods 2.1. Participants The study started with 12 black belt male athletes from the Iranian National Taek- wondo League, but four taekwondo athletes could not complete our study due to injury during the period of the investigation. Therefore, eight athletes (age: 20 4 years; height: 180 2 cm; body mass: 64.8 4.0 kg) completed the study. All athletes were from the same club to prevent potential interference/variation caused by different training programs implemented among taekwondo clubs. The inclusion criteria included: (1) more than ve years of experience in taekwondo; (2) not having consumed any ergogenic aids including: creatine, beta-alanine, caffeine, sodium bicarbonate, and caffeine content supplements three months before the study; (3) be training at least ve times a week; (4) be older than 18 years old; (5) not currently having any musculoskeletal injuries. This study was conducted dur- ing the 4-week pre-competition preparation phase. During this time, participants trained six sessions per week, including three taekwondo-speci c training and three conditioning sessions, including strength training and taekwondo-speci c tness. The Sport Science Research Institute of Iran approved of current study (IR.SSRC.REC.1399.062). 2.2. Experimental Design This study had a randomized, double-blind placebo-controlled crossover design with ve testing sessions at the laboratory. The rst session was used to explain the experimental procedures, take anthropometric measurements, and familiarize with the Multiple Frequency

three conditioning sessions, including strength training and taekwondo-speci c tness. The Sport Science Research Institute of Iran approved of current study (IR.SSRC.REC.1399.062). 2.2. Experimental Design This study had a randomized, double-blind placebo-controlled crossover design with ve testing sessions at the laboratory. The rst session was used to explain the experimental procedures, take anthropometric measurements, and familiarize with the Multiple Frequency Speed of Kick Test (FSKT), countermovement jump (CMJ), and the Progressive Speci c Taekwondo Test (PSTT). All participants were familiar with the above speci c tests, but under supervision of a quali ed coach, the participants repeated the tests until the coach considered the technique was appropriate. Subsequently, participants were set to 4 conditions: (1) 60 mL BJ (400 mg NO3 ) + 60 mL placebo (PL) (BJ-400), (2) 60 mL BJ + 60 mL BJ (BJ-800), (3) 60 mL PL + 60 mL PL (PL), and (4) control (CON). The washout between sessions was seven days, and all measurements were performed between 10:00 a.m. and 4:30 p.m. at the same day of each week to align with normal weekly routines [34]. Participants were instructed to prevent strenuous exercise 24 h before each session. To prevent overreaching, coaches were requested to control the training volume and intensity throughout the study. The Hooper Index questionnaire was used before each test to monitor and evaluate the recovery and accumulated fatigue [34,35]. The experimental design is summarized in Figure.

Int. J. Environ. Res. Public Health2021,18, 10202 4 of 17Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 4 of 18 Figure 1. Schematic representation of the experimental design. BJ, beetroot juice; CMJ, counter movement jump; FSKT, Frequency Speed of Kick Test; PSTT, Progressive Specific Taekwondo Test; RPE, rating of perceived exertion. 2.3. Supplementation Protocol and Standardization of Physical Activity and Diet Participants arrived for the four trials (BJ-400, BJ-800, PL, CON) at the laboratory 3 h prior to the tests. Two hours and thirty minutes before starting the tests, each participant ingested either one bottle of 60 mL BJ + one bottle of 60 mL of PL or two bottles of BJ (120 mL) (Red Beet Vinitrox Shot; Sponsor Ltd.; Germany) containing 400 mg NO 3 − per bottle or depleted dried powder NO 3 − for PL [36]. Based on previous instructions, PL was pre- pared by dissolving 1 g of dried powder of BJ into one liter of water and adding lemon juice to mirror the taste of the commercial supplement [36]. Both BJ and PL were provided in identical bottles. Based on the pharmacodynamics of NO 2 − after BJ ingestion [37] and the recommendation of ingesting BJ 2.5–3 h before exercise to synchronize with the peak of plasma NO 2 − [11,29,38], supplementation was given 2.5 h previous to FSKT. In consid- eration of the anti-bacterial effect in the mouth by oral antiseptics which may prevent the rise in blood NO 2 − after the intake of NO3 −, participants were instructed to abstain from teeth brushing, using mouthwash, chewing gum, or sweets that could contain anti-bacte- rial substance during the 24 h prior to the test session [36]. Furthermore, a list of dietary sources rich in NO 3 − was given to participants, with instructions to avoid eating those in the 24 h prior to arriving at the laboratory (i.e., beetroot, celery, arugula, lettuce, spinach, turnip, endives, leek, parsley, cabbage, etc.). Participants were also instructed to avoid drinks containing caffeine and nitrate-rich fruits and vegetables 72 h before the tests due

of dietary sources rich in NO 3 − was given to participants, with instructions to avoid eating those in the 24 h prior to arriving at the laboratory (i.e., beetroot, celery, arugula, lettuce, spinach, turnip, endives, leek, parsley, cabbage, etc.). Participants were also instructed to avoid drinks containing caffeine and nitrate-rich fruits and vegetables 72 h before the tests due to its potential ergogenic effect. Participants were asked to replicate their food and drink intake 24 h before each session. On the test day, water was provided ad libitum during the rest period between the tests, and a standardized snack was ingested four hours before starting the test consisted of 1.5 g/kg carbohydrate and 20 g protein [35]. 2.4. Frequency Speed of Kick Test Multiple The FSKT included 5 sets of FSKT, and each set’s period was 10 s interspersed with 10 s passive rest. Each athlete had to stay in front of the punching mitt to perform the test. After the sound signal, athletes performed the maximal number of kicks, alternating right and left legs. Performance was measured via the number of kicks in each set, the total number of kicks, and the kick decrement index (KDI) during the test. KDI indicates the Figure 1. Schematic representation of the experimental design. BJ, beetroot juice; CMJ, counter movement jump; FSKT, Frequency Speed of Kick Test; PSTT, Progressive Speci c Taekwondo Test; RPE, rating of perceived exertion. 2.3. Supplementation Protocol and Standardization of Physical Activity and Diet Participants arrived for the four trials (BJ-400, BJ-800, PL, CON) at the laboratory 3 h prior to the tests. Two hours and thirty minutes before starting the tests, each participant ingested either one bottle of 60 mL BJ + one bottle of 60 mL of PL or two bottles of BJ (120 mL) (Red Beet Vinitrox Shot; Sponsor Ltd.; Germany) containing 400 mg NO3 per bottle or depleted dried powder NO3 for PL [36]. Based on previous instructions, PL was prepared by dissolving 1 g of dried powder of BJ into one liter of water and adding lemon juice to mirror the taste of

of PL or two bottles of BJ (120 mL) (Red Beet Vinitrox Shot; Sponsor Ltd.; Germany) containing 400 mg NO3 per bottle or depleted dried powder NO3 for PL [36]. Based on previous instructions, PL was prepared by dissolving 1 g of dried powder of BJ into one liter of water and adding lemon juice to mirror the taste of the commercial supplement [36]. Both BJ and PL were provided in identical bottles. Based on the pharmacodynamics of NO2 after BJ ingestion [37] and the recommendation of ingesting BJ 2.5–3 h before exercise to synchronize with the peak of plasma NO2 [11,29,38], supplementation was given 2.5 h previous to FSKT. In consideration of the anti-bacterial effect in the mouth by oral antiseptics which may prevent the rise in blood NO2 after the intake of NO3 , participants were instructed to abstain from teeth brushing, using mouthwash, chewing gum, or sweets that could contain anti- bacterial substance during the 24 h prior to the test session [36]. Furthermore, a list of dietary sources rich in NO3 was given to participants, with instructions to avoid eating those in the 24 h prior to arriving at the laboratory (i.e., beetroot, celery, arugula, lettuce, spinach, turnip, endives, leek, parsley, cabbage, etc.). Participants were also instructed to avoid drinks containing caffeine and nitrate-rich fruits and vegetables 72 h before the tests due to its potential ergogenic effect. Participants were asked to replicate their food and drink intake 24 h before each session. On the test day, water was provided ad libitum during the rest period between the tests, and a standardized snack was ingested four hours before starting the test consisted of 1.5 g/kg carbohydrate and 20 g protein [35]. 2.4. Frequency Speed of Kick Test Multiple The FSKT included 5 sets of FSKT, and each set's period was 10 s interspersed with 10 s passive rest. Each athlete had to stay in front of the punching mitt to perform the test. After the sound signal, athletes performed the maximal number of kicks, alternating right and left legs. Performance was measured via the number of

Test Multiple The FSKT included 5 sets of FSKT, and each set's period was 10 s interspersed with 10 s passive rest. Each athlete had to stay in front of the punching mitt to perform the test. After the sound signal, athletes performed the maximal number of kicks, alternating right and left legs. Performance was measured via the number of kicks in each set, the total number of kicks, and the kick decrement index (KDI) during the test. KDI indicates the

This study investigates the effects of beetroot juice on taekwondo performance and cognitive function.