Participants
Twelve recreationally-trained males participated in the three-week investigation (mean +/- SD, age: 28 +/- 5 y, height: 178 +/- 9 cm, weight: 79.15 +/- 15.7 kg, VO2max: 45.7 +/- 7.6 ml/kg/min). The investigation was approved by an Institutional Review Board for use of human subjects, and all subjects signed an informed consent and completed a health history questionnaire prior to the investigation. All subjects met the necessary inclusion requirements as assessed by a health history and exercise questionnaire. Subjects were required to have been free of any nutritional supplements or ergogenic aids for at least 6 weeks preceding the study and were asked to refrain from using any additional supplements during the course of the study. In addition, subjects regularly engaged in exercise activity on a consistent basis (workout days per week: 3.9 +/- 1.3 days, workout hours per day: 1.7 +/- 1 h, cardio training per week: 3.8 +/-1.8 h, resistance training per week: 4.3 +/- 1.8 h). Subjects also reported participation in one or more of the following sport activities: running, weightlifting, swimming, cycling, tennis, baseball, basketball, football, kickboxing, lacrosse, and mixed martial arts.
Study design
This study used a randomized, double-blind, placebo-controlled cross-over design. Subjects reported to the human performance laboratory on three separate occasions. All participants completed a baseline/familiarization day of testing, including a maximal graded exercise test for the determination of aerobic capacity (VO2max), one-rep maximum (1RM) for bench and leg press to determine 75% of 1RM, choice reaction familiarization, which included single-step visual and audio protocol, one tower protocol, two tower lateral protocol, three tower multi-directional, three tower multi-direction protocol with martial arts stick, and intermittent critical velocity familiarization completed at 110%, 100%, and 90% of VO2max. Subjects were instructed to refrain from performing any strenuous activity for 24 h before the subsequent testing sessions (Visit 2, 3). Subjects were also required to maintain their current training program throughout the duration of the study. Additionally, subjects were required to complete a 2-day food log before the second testing day. Two days prior to Visit 3, subjects were asked to consume the exact same foods/beverages eaten and written in their 2-day food log as recorded prior to Visit 2. Subjects reported to the lab in a fasted state (12 h with ad libitum water) to ensure that any additional foods/beverages would not influence performance or negate the potential effects of the supplement (SUP) or placebo (PL). Following familiarization, subjects were randomly assigned to ingest either the SUP or PL for Visit 2 and were provided the cross-over treatment on the last testing visit. Twenty minutes after ingestion, blood pressure and heart rate (HR) were re-assessed, and subjects completed a four-question survey (5-point likert scale) that asked them to describe their feelings of energy, fatigue, alertness, and focus at that particular moment [1]. Subjects were asked to rate their energy level, fatigue level, feelings of alertness, and feelings of focus using the following verbal anchors: 1 = very low; 2 = low; 3 = average; 4 = high; 5 = very high. The questionnaire was administered immediately before each choice reaction testing session (Figure 1). Subjects ingested each supplement 20 min prior to testing since that is what is typically practiced by most recreational athletes.
Choice reaction test
Reaction time was measured using the Makoto II Arena testing device (Makoto USA, Centennial, CO, USA). The Makoto II Arena device is constructed in the shape of a triangle that is 2.44 m from base to base and consists of three steel towers that are 1.83 m high with each tower containing 12 targets (Figure 2). Most studies that have investigated choice reaction time failed to utilize a testing device and protocol that is related to sport-specific training and team-sport performance. Due to the fact that most team sports require quick agile movements in different directions, the current investigation sought to utilize a testing device and protocol that measured stationary, lateral, and multi-directional movements. Each subject underwent nine different tests, measuring choice reaction time for each test. First, subjects were tested on a two-tower single step protocol. The reaction test consisted of audio (CRA) and visual (CRV) protocols, each completed separately. The decision to separate reaction time cues was made to accommodate the physiological transduction rate disparities between auditory and visual stimuli [13]. Targets used for this protocol included the middle target on each tower, located approximately at chest-level. The audio protocol consisted of only an auditory stimulus; therefore, a magnetic cover with the label (X) was used to cover the specified target on each tower. The visual protocol consisted of only visual alerts, in which illumination of selected bulbs provided the stimulus. The volume was muted during this test, allowing subjects to use only their visual sense to detect which target illuminated. For each test, subjects stood in the middle of the triangle behind a marked line located 1.55 m from both towers. Subjects stood behind the line with the third target (not being used) directly behind them (Figure 2). Subjects were required to lunge and make contact between their hand and the specified target on either tower. Built-in computer software randomly displayed the target on one of the two towers in the equivalent location.
Reaction time for each subject was calculated using built-in software and was recorded for each trial. Randomization was used to determine whether the subject would complete either the audio or visual protocol first. Subjects repeated each test for a total of three attempts, and the mean of the last two trials was used in analysis. Subjects then completed testing protocols at durations of 15 s, using both audio and visual alerts. Testing procedures included one tower, for which the subjects remained stationary and only used their hands (CRS15), followed by two towers starting from the same line as CRV and CRA, for which subjects moved laterally from side to side (CRL15), and then three towers, for which subjects moved in multiple directions (CRM15); with two attempts for each test. The same testing procedures were followed again; only this time the duration was 30 s (CRS30, CRL30, CRM30). Mean reaction time for each trial was calculated internal to the device and recorded. The average of two back-to-back attempts was calculated and used for analysis. Skill levels indicated how long each target remained illuminated: the lower the level, the longer the target was lit. Each of the aforementioned tests was completed at the first skill level (level 1, 3 s count) to ensure each subject's ability to register a 'hit' with ease. Test-retest reliability of the above protocol calculated from six men at least 2 days apart yielded an ICC > 0.94 and a CV less than 10% for all choice reaction tests. Subjects completed the choice reaction testing protocol with a (three-tower) protocol using a large martial arts stick for two minutes. This protocol was set at the fourth skill level (1.20s count) to mimic the protocol used in a similar study by Hoffman et al. [1]. In our laboratory, test-retest reliability of the above protocol from six men at least 2 days apart yielded an ICC > 0.81. Average reaction time tested at different durations and different types of movements (stationary, lateral, multi-directional) allowed for a comprehensive analysis of agility compared to standard agility tests, such as the pro-shuttle and t-test for which agility speed, and not reaction time, is measured.
Determination of VO2max
All VO2max tests were performed on a calibrated treadmill (Woodway Desmo, Waukesha, WI) and measured by indirect calorimetry using a metabolic cart (Parvomedics True One 2300, Sandy, UT, USA). Testing began with a three-minute warm-up stage at 5.15 km/h, while the grade remained at 0% throughout testing. Velocity increased to 10 km/h for the next stage, then increased 2 km/h for every two minutes up to 16 km/h, followed by 1 km/h increments per minute up to 24 km/h. VO2max was confirmed if at least two of the following criteria were met: (a) plateau in HR or HR values within 10% of the age-predicted HRmax, (b) plateau in VO2 (defined by an increase of no more than 150 ml·min-1), (c) respiratory exchange ratio (RER) value greater than 1.15.
Intermittent critical velocity and intermittent anaerobic running capacity
Intermittent Critical Velocity (iCV) testing protocols were similar to previous studies [2, 14]. All iCV tests were performed on a calibrated treadmill (Woodway Desmo, Waukesha, WI, USA). Subjects completed three separate treadmill running tests at intervals of 10 s of running and 10 s of rest at 110%, 100%, and 90% of their peak velocity at VO2max. A 25-minute break period was given between running tests. During this break period, subjects performed the choice reaction protocol followed by approximately five minutes of inactivity before the next treadmill test. Each iCV testing session was terminated when the subject could no longer complete a 10s sprint. Distance and total running time were recorded for each participant, and iCV and intermittent anaerobic running capacity (iARC) were calculated using the following equation: (Total Distance = iARC + iCV · total running time). An investigation by Fukuda et al., 2009 [14] examined the reliability of iCV testing in men and women, producing an ICC value of 0.87 and 0.83.
Muscular strength and endurance
One-repetition maximum bench press and leg press were used to determine the maximum amount of weight a subject could lift one time for each exercise. Subjects performed one warm-up set for bench and leg press (≈50% of estimated 1RM) and rested for two minutes between attempts. Resistance was increased until the participant failed to complete a repetition; subjects performed no more than five attempts. Maximum strength for bench and leg press exercises was measured using plate-loaded iso-lateral bench and leg press (Hammer Strength, Cincinnati, OH, USA). Seat height for bench and leg press was adjusted for each subject and remained consistent throughout the course of the study. A repetition was recorded when subjects lowered the weight in an eccentric contraction and then pushed the weight in a concentric contraction with full joint extension, ensuring the weights made contact with the back bumper pads after eccentric movement. Following 1RM testing, 75% of each subject's 1RM was calculated, and repetitions to fatigue were assessed and recorded at respective weight resistance on subsequent testing visits. Subjects were required to perform a set (at least 5 repetitions) of 75% of their 1RM for familiarization on Visit 1.
Supplement
On the second and third testing days, subjects consumed 1 scoop (2 servings, 46 g) of either the PL or SUP powder mixed with ≈ 16 oz. of water. The SUP, marketed as Assault™ (MusclePharm, Denver, Colorado, USA), contained BCAAs (6 g), creatine (5 g), beta-alanine (4 g), citruline malate (1.5 g), and caffeine (300 mg). Complete nutritional facts are listed in Figure 3. The PL was flavored maltodextrin, similar in color, taste and flavor to the SUP formulation. An investigator secluded from any data collection prepared and administered both supplement and placebo beverages for all subjects, using the same amount of water and powder for each beverage. Subjects were provided cold water ad libitum throughout the study.
Data analysis
All recurring tests were analyzed using a repeated measure analysis of variance (ANOVA) [time (T1-T4) × drink (SUP vs. PL)]. Significant interactions between time and supplement were analyzed using post-hoc dependent t-tests. In addition, iCV and iARC were analyzed using dependent t-tests. Significant main effects for time and for supplement were analyzed using Bonferroni post-hoc tests to account for multiple comparisons by maintaining family-wise error rates. A p-value less than 0.05 was considered significant for this investigation. All ANOVA assumptions were met, and analyses were performed using the computer program SPSS (PASW Statistics 18.0.0, IBM Corporation, Armonk, NY, USA).