Participants
Twelve male taekwondo athletes were recruited from National Taiwan University of Sport, Taichung, Taiwan. All subjects have been participating in taekwondo training for at least 6 years and competed at the national or international level. The subjects have the age of 20.0 ± 0.8 years, the height of 1.77 ± 0.04 m, the weight of 66.9 ± 5.0 kg, the body mass index of 21.29 ± 0.93 kg/m2, and VO2max of 44.9 ± 6.8 ml/min/kg. The exclusion criteria included cardiovascular disease risks, musculoskeletal injuries, smoking, consumption of protein supplement or under any medication in the past 3 months. The regular training schedule and diet habits were maintained during the study period. The subjects were refrained from all training activity on the day prior to the trial. All subjects gave their written informed consent after the experimental procedure and potential risks were explained. The study protocol was approved by the Research Ethics Committee of China Medical University and Hospital.
Experimental design
This study used a double-blind, randomized cross-over design (Fig. 1). Each subject completed amino acids (AA) and placebo (PL) trials in a random order, separated by a wash-out period of at least 7 days. The same food, purchased from local convenience stores, was provided during the 2 days prior to the trials. The meals provided approximately 1800 kcal/day with 54 % energy from carbohydrate, 30 % from fat, and 16 % from protein, according to the manufacturer’s label. The breakfast on the days of trials included white bread 1.2 g/kg, jam 0.1 g/kg, butter 0.l g/kg, and soybean milk 5 ml/kg (6.2 kcal/kg, containing carbohydrate 1.0 g/kg, protein 0.24 g/kg, and fat 0.14 g/kg) [24].
Experimental procedure
Approximately 1–2 weeks prior to the first trial, cardiopulmonary function was measured. The subjects warmed up on an electrically braked cycle ergometer (Corival, Lode, Groningen, Netherland) at 50 W for 5 min, followed by incremental stages of 25 W every 3 min. The breath-by-breath gas was analyzed (Vmax 29C, Sensormedics, Yorba Linda, CA, USA). Maximal oxygen uptake (\( \overset{.}{\mathrm{V}}{\mathrm{O}}_{2 \max } \)) was considered to be achieved when the subjects were unable to maintain the workload.
Supplementation
On the days of the trials, the subjects reported to the laboratory at 0700 after an overnight fast. After collecting venous blood samples as the baseline, the subjects consumed the standardized breakfast. Each trial included 3 simulated matches. After the second match, 2 supplementations were consumed. In the AA trial, the subjects ingested 0.17 g/kg BCAA (leucine: isoleucine: valine = 10:7:3, containing vitamin E 6.67 IU/g BCAA, capsule, General Nutrition Corporation, Pittsburgh, PA, USA), 0.05 g/kg arginine and 0.05 g/kg citrulline (arginine: citrulline = 1:1, tablet, General Nutrition Corporation). In the PL trial, the subjects consumed the identical amount of empty capsule and tablet containing starch (Chung-Yu Biotech Co LTD, Taichung, Taiwan) to the AA trial and 1 capsule of vitamin E (100 IU, General Nutrition Corporation). All supplements were taken with water within 10 min. Our preliminary study has shown that plasma BCAA and arginine concentrations would peak after 1 h of ingestion (data not shown). Therefore, the supplements were consumed 1 h before the third simulated match in this study. In addition, the supplements were given prior to the third match because our pilot study showed that cognitive function started to decline after 2 simulated matches (data not shown).
The subjects were allowed to drink water ad libitum in the first trial, then the timing and amount of consumption were repeated in the following trial. The water consumption was 1133.3 ± 548.6 mL in both trials.
Simulated match
The simulated match was designed to mimic the high-intensity intermittent nature of actual taekwondo competitions, modified from the previous study [31]. The exercise was performed on a cycle ergometer (894E, Monark, Varberg, Sweden). Each trial contained 3 matches with a 1-hr rest before the second match and a 2-hr rest before the third match. A match included three 2-min rounds with repeated work to rest time of 5 s and 25 s, respectively. A 1-min rest was provided between the rounds. The work to rest ratio of 1 to 5 was determined according to the analysis of international taekwondo matches [32]. During the working period, the load was set at 0.1 kp/kg body weight. The subjects were asked to pedal as fast as possible while the research personnel providing vocal encouragement. The peak and average power of each 5-s sprint was recorded. During within-round and between-round rest periods, the subjects pedaled at 60 rpm without the load.
Reaction test battery
This taekwondo-specific reaction battery, containing 3 single- and 1 dual-task movements, has been shown to exhibit moderate to high reliability and validity in elite and sub-elite athletes. The intraclass correlation coefficients were 0.439–0.634 in PRT in single-task movements, and 0.692 in reaction time in the secondary task in elite taekwondo athletes [29]. Each of the 4 movements was performed 5 times in a random order. A researcher told the subject which movement was to be performed prior to each task. All subjects were right-handed and performed the kicks with their right leg. Electromyography (EMG) electrodes were attached to left thenar and brachioradialis muscles.
The detailed procedure in this battery can be found elsewhere [29]. Briefly, the subjects stood in a guard position with both heels on a force platform (9260AA6, Kistler, Winterthur, Switzerland) while holding a button on the left hand. The subjects were asked to press the button on the left hand with the thumb as soon as they see a light signal from the top of the head of a dummy, then start the respective movement to attack the dummy. Three sets of single-task movements were used: (A) a roundhouse kick to the rib; (B) a roundhouse kick to the rib, a roundhouse kick to the head, then a reverse roundhouse kick to the head; and (C) a roundhouse kick to the rib, a roundhouse kick to the head, a reverse roundhouse kick to the head, a roundhouse kick to the head, a reverse roundhouse kick to the head, then a roundhouse kick to the head. The subjects can only put the right foot back to the ground after all kicks were performed in the movement. The signals from EMG, force platforms, the button, and the accelerometer (EGAXT3; Measurement Specialties, Hampton, VA, USA) in the dummy were collected through a data acquisition and analysis system (MP150, Biopac Systems, Inc., Goleta, CA, USA).
The dual-task movement D is composed of movement C, the primary task, and a secondary task. While the subjects were carrying out the primary task, research personnel turned on the light signal on the dummy again. The subjects then press the button with their left thumb as soon as they see the second light signal.
In single-task tests, PRT was determined as the time from the beginning of the light signal to the start of EMG signal of the left thenar muscles. The thenar muscles were used because they provided much clearer EMG signal compared to the leg muscles. The EMG signal from leg muscles was noisy due to the unconscious preparation for the kicks before the light, even though the subjects were asked to stand still prior to the stimulus. Motor reaction time was between the start of EMG signal of the left thenar muscles and the right leg leaving the force platform. Movement time was between the right leg leaving the force platform and the appearance of signal from the accelerometer in the dummy.
In the dual-task test, the performance of the secondary task was defined as the time between the beginning of the second light signal and pushing the button. Therefore, the performance of the secondary task involves PRT, motor reaction time, and movement time. EMG signal was not used in measuring the secondary task because it was present throughout the first task. Therefore, it was very difficult to identify the EMG signal that triggered the movement to press the button.
Blood sample collection
Venous blood samples were collected before breakfast, immediately after each match, and immediately before the start of the third match. At each sampling time, a 16 ml blood sample was collected into a tube containing EDTA. The blood samples were centrifuged at 1500 x g (Eppendorf 5810, Hamburg, Germany) to extract plasma. The aliquoted plasma samples were stored at −70 °C until further analysis.
Measurement of blood biochemical parameters
Plasma BCAA concentration was measured enzymatically (Biovision, Milpitas, CA, USA). The absorbance at 450 nm was measured with a microplate spectrophotometer (Benchmark Plus, Bio-Rad, Hercules, CA, USA). Plasma free tryptophan concentration was analyzed with a fluorescence assay (Bridge-It, Mediomics, St. Louis, MO, USA). The fluorescence at excitation 485 nm and emission 665 nm was read by a microplate fluorescence reader (Plate Chameleon, Hidex, Turku, Finland). Plasma NOx concentrations were determined using Griess reagent [33] and the absorbance at 450 nm was measured with a microplate spectrophotometer. Plasma concentrations of urea, glucose, lactate, NH3, glycerol, and NEFA were measured with an automatic analyzer (Hitachi 7020, Tokyo, Japan) using commercial kits (Randox, Antrim, UK). Plasma concentrations of all parameters were corrected for the changes in plasma volume using hemoglobin concentration and hematocrit in whole blood [34].
Statistical analysis
All values were expressed as mean ± SD. The results were analyzed by 2-way (trial x time) analysis of variance with repeated measurements. If the time x trial interaction effect is significant, the difference between the 2 trials after the third simulated match was identified by one-way analysis of covariance with the pre-exercise level as the covariant. If the time effect is significant, the differences between each time points within the same trial were determined by post hoc Bonferroni analysis. A p-value less than .05 was considered statistically significant.
Availability of data and materials
The dataset supporting the conclusions of this article is available in ResearchGate (https://www.researchgate.net/publication/303405818_Branched-chain_amino_acids_arginine_citrulline_alleviate_central_fatigue_after_3_simulated_matches_in_taekwondo_athletes_a_randomized_controlled_trial).