This study was a randomized, double-blind, placebo-controlled crossover trial. After familiarization, each participant completed two testing sessions that were identical with the exception of the provided supplement. On one occasion, a dietary supplement (SUP) containing proprietary blend of caffeine, theanine and tyrosine was provided. The SUP contained no other ingredients and only those previously listed. During the other testing session, a placebo (PL) containing ~ 2 g of maltodextrin was provided. Within each testing session, participants completed assessments of mental and physical performance before and after ingestion of the supplement, as well as after two rounds of exercise.
Participants were recruited from the university student population. In order to be eligible, individuals were required to meet the following inclusion criteria: 1) male between the ages of 18 and 25; 2) current or former collegiate athlete (all NCAA Division 3); 3) non-smoker; 4) healthy and disease-free; 5) regular caffeine consumer; and 6) willingness to comply with study protocol. To quantify caffeine consumption, each participant completed a caffeine questionnaire identifying daily habitual caffeine intake. Additionally, each participant completed a health history questionnaire and the Physical Activity Readiness Questionnaire (PAR-Q). Individuals were ineligible for participation if they: 1) had not been engaged in regular exercise training for at least 12 months prior to the study; 2) answered “yes” to any question on PAR-Q; 3) were diagnosed with any disease or were currently taking prescription medications; 4) were currently using a dietary supplement other than a multi-vitamin/mineral, protein powder or meal replacement, or had used such supplements in the past 6 weeks; 5) were currently enrolled in another research study or had been enrolled in the past 8 weeks; or 6) had any known allergy or sensitivity to caffeine or other stimulants, as determined by the health history questionnaire. Each participant also completed an adverse events questionnaire prior to and at the end of each testing session. Questions included, but were not limited to, symptoms of gastrointestinal distress, headaches, and dizziness. A university-approved informed consent document was signed by each participant prior to study commencement. This study was approved by the Institutional Review Board at the University of Mary Hardin-Baylor.
After screening and informed consent, the height and weight of each participant was obtained using standard procedures. Body composition of each participant was assessed via multi-frequency bioelectrical impedance analysis (InBody 770, Seoul, Korea). Participants were also familiarized with the performance testing device used in this study (Makoto Arena, Makoto USA, Illinois). This device evaluates reaction time, cognitive function and overall mental and physical performance during specific tasks. This performance testing device consists of three towers, each with lights located at the base as well as at other locations on the tower. During testing, audio is associated with flashing lights with higher pitch noises corresponding to light targets at a higher vertical placement on the tower and lower pitch noises corresponding to targets at the base of the towers. Lights on the left side of the towers were required to be hit by the participant’s left hand, while lights on the right side of the tower were required to be hit by the participant’s right hand and lights in the middle of the tower could be hit with either hand. Lights at the base of the tower were required to be hit by the participant’s feet. During one-tower testing, participants remained adjacent to a single tower during the 30-s test, which was performed on level 11 (i.e. the fastest setting). During three-tower testing, participants were required to move about the arena in order to hit lights produced by any of the three towers. The three-tower testing was performed on level 6 (i.e. a mid-range speed). The specific variables assessed by the performance testing system were the total targets, targets hit, average hit time and accuracy. During familiarization, participants completed trials on the device until they had less than a 10% difference between consecutive scores for targets hit. On average, this took approximately 6 trials. Following familiarization, participants were randomly assigned to an order in which to complete the two testing sessions.
Participants provided dietary records for the 3 days prior to each testing session. Additionally, they were required to abstain from unfamiliar exercise and exercise of greater-than-usual intensity for 48 h prior to each session, as well as abstain from all exercise for 24 h prior to each session. Each participant was allowed an ad libitum breakfast on the day of testing, followed by a standardized lunch (MET-Rx® Big 100 meal replacement bar; 400 kcal, 48 g carbohydrate, 10 g fat, 31 g protein) that was consumed 2 to 3 h prior to the acute testing session. Water was the only beverage allowed on the day of testing, and participants were instructed to discontinue water consumption one hour prior to testing. Caffeine intake was disallowed 24 h prior to and on the day of testing. At the beginning of each session, pre-supplementation (T1) assessments took place. These included evaluations of body weight, resting hemodynamic variables (i.e. heart rate and blood pressure), standard visual analog scales (VAS) to quantify subjective variables (i.e. energy, focus, concentration, alertness, fatigue and motivation) and testing on the Makoto performance assessment device. After these tests were completed, each participant ingested the assigned dietary supplement (i.e. SUP or PL) with 8 oz of water under researcher supervision then rested quietly for 30 min. Following this rest period, the VAS and Makoto assessments were repeated (T2) before each participant completed one round of exercise. The round of exercise consisted of nine individual exercises, each of which were performed continuously for 45 s with 10 s of rest between exercises. The individual exercises included were: small battle rope waves, large battle rope waves, battle rope slams, kettlebell swings, line jumps, toe touches, mountain climbers, bosu squats and burpees. Physical performance during each round of exercise was quantified via the amount of reps completed per exercise per set, and heart rate was monitored before and after each round of exercise and Makoto assessment. Upon completion of the first round of exercise, the VAS and Makoto assessments were repeated (T3), with the Makoto assessments commencing 90 s after the completion of the exercise round. This was followed by a second identical round of exercise and final VAS and Makoto assessments (T4). At the end of the final one- and three-tower Makoto assessments, the Makoto infinity test was performed. This testing program continuously generated a random sequence of visual targets and auditory signals which prompted the participant to hit the specified targets. The infinity test continued until the participant missed 3 total targets. In addition to the performance testing procedures, each participant completed a side effects questionnaire.
Following the completion of the first condition, participants entered a 1-week washout period with instructions to maintain habitual exercise and nutritional routines. Participants adhered to the same pre-assessment procedures and completed an additional 3-day diet record prior to the second condition. The second condition was identical to the first, with the exception of which dietary supplement was provided. Additionally, the second condition occurred on the same day of the week and time as the first condition. For both conditions, participants were not scheduled on “high stress” days, including those with examinations or athletic competitions.
General linear models were used to test for the effects of the dietary supplement condition, time, and their interactions on mental and physical performance. Data were transformed when skewed distributions were present. Change scores, where applicable, were generated by subtracting the baseline values from the values at each subsequent time point (i.e. T2, T3 and T4). Non-parametric Kruskal-Wallis tests were conducted for measures with skewed distribution to compare the differences cross treatment groups at each time point. Paired samples t-tests were used to compare dietary intake, heart rate and blood pressure prior to each session, and general linear models were used to evaluate heart rate responses to exercise. Statistical significance was set at P < 0.05. Analyses were performed using Minitab 17 (Minitab Inc., State Collage, PA) and SPSS 25 (IBM, Armonk, NY).