Pre-workout supplementation has become a fundamental component in nutrition programs and a growing interest in the sports nutrition industry. Recent research indicates energy drinks are the most popular supplement next to multi-vitamins in the young adult population (18-35 year) [1]. Many athletes believe supplementation prior to training will result in greater focus, quicker reaction time, and increased power [1]. Performance-enhancing claims of dietary supplements have not been fully addressed in the context of sport-specific exercises. Therefore, examining the effects of nutritional supplements during an exercise training session has the potential to elucidate more practical recommendations and applications [2].

Most supplements contain different ingredients to produce ergogenic effects. When ingested together, these ingredients may work synergistically to enhance various aspects of exercise performance. The ergogenic effects of caffeine have been attributed to a number of possible mechanisms, primarily the blocking of adenosine receptors [3, 4]. Caffeine inhibits action at adenosine receptors, which has been reported to decrease the perception of pain and effort, resulting in improved exercise performance [5, 6]. Research has also suggested that adenosine-receptor antagonism contributes to improved performance via increases in neurotransmitter release and motor firing rate [3, 7]. However, the effects of caffeine in combination with beta-alanine, branched chain amino acids (BCAAs), creatine, vitamin B-6, and vitamin B-12 on ratings of perceived effort and pain have not been well investigated, particularly when ingested as a pre-workout supplement. Both beta-alanine and creatine have been shown to delay the onset of neuromuscular fatigue and therefore potentially augment the ergogenic effect of caffeine [8, 9]. Research has shown vitamin B-6 to play an important role in the metabolic pathways required for exercise [10], while vitamin B-12 assists with DNA synthesis, which is necessary for the formation of red blood cells [10]. Research suggests that when supplementing with BCAAs prior to physical activity, recovery improves due to an increase in protein synthesis and a reduction in protein degradation [11]. However, studies have indicated that BCAAs do not improve endurance performance [12]; therefore, further research is required to examine the effects of BCAA supplementation on various team sport-related performances (e.g. repeated sprint ability) [3].

Few studies have examined the effect of pre-workout supplements using protocols related to sports-specific training and team sport performance. Therefore, the purpose of this study was to determine the effects of a supplement containing BCAAs, creatine, beta-alanine, citruline malate, arginine, vitamin B-6, vitamin B-12, and caffeine (Assault™, MusclePharm, Inc., Denver, CO, USA) on upper and lower body muscular endurance, aerobic and anaerobic capacity, and choice reaction time.

This investigation was the first to examine the effects of the pre-workout supplement Assault™ on a multi-faceted, exercise testing protocol. The results suggest that the pre-workout supplement significantly improved muscular endurance and choice reaction time.

The primary active ingredient in Assault™ is caffeine. Caffeine is a mild stimulant that affects the central nervous system and has the potential to influence human neuromuscular performance. In an attempt to maximize the effectiveness of caffeine, supplement manufacturers often combine several ingredients, possibly enhancing caffeine's stimulatory potential [1]. A study conducted by Fukuda et al. [15], showed an acute anaerobic effect in men and women with the ingestion of a low dosage of caffeine, BCAAs, and creatine, which could possibly lead to improvements in various aspects of human performance (muscle strength, power, etc).

In the current investigation, a 26.5% reduction in total repetitions (3 reps) at 75% of 1RM was observed from time point 1 to time point 2 in the PL group for leg press, while an 11.7% (2 reps) reduction from time point 1 to time point 2 was observed in the SUP treatment for leg press. Results also indicated positive effects on perceived alertness, energy, and focus, which may have contributed to a 4% improvement in CRA and CRM30, and a 5% improvement in CRM15. These findings are consistent with previous studies [16, 17] that also reported significant improvements in choice reaction time following the ingestion of a supplement containing 5-6 mg/kg of caffeine, which is higher than the amount used in the current study (mean = 3.8 +/- 0.7 mg/kg).

There are many possible reasons for the improvements in performance demonstrated in this study. At the cellular level, caffeine enhances neuromuscular transmission and improves skeletal muscle contractility [18]. Green et al. [19] investigated the effects of caffeine (6 mg/kg) on lower body muscular endurance with repetitions performed to volitional failure. Results indicated that caffeine was associated with significantly higher repetitions (12.5 ± 4.2, 9.9 ± 2.6) during the third set of leg press exercises compared to PL [19]. In agreement, the current investigation showed significant increases in both total leg press repetitions (SUP 13 ± 6, PL 11 ± 3, p = 0.021) at T1 and T2 (SUP 12 ± 5, PL 8 ± 6, p = 0.024), although no significant time and SUP interaction was found (Figure 4).

A study by Barry et al. [20] examining caffeine-induced arousal effects on performance and auditory event-related potentials in the brain, indicated a reduction in reaction time and suggested that caffeine differentially improves processing aspects related to task performance [20]. The decrease in CRA times may have had an effect on the improvement in CRM15 and CRM30 because they also include an auditory component. Figure 5 illustrates that the reduction in multi-directional choice reaction time (CRM) for 15 s and 30 s was most likely due to the significant decrease in choice reaction time using an auditory stimulus (CRM15, SUP: 1.07 ± 0.12 s, PL: 1.13 ± 0.14 s, p = 0.007; CRM30, SUP: 1.10 ± 0.11 s, PL: 1.14 ± 0.13 s, p = 0.013). Caffeine's stimulatory effects on the central nervous system seem to manifest in performance in activities which require quick reactions and movements [18]. Thus, it is evident that choice reaction time improved following the ingestion of 3.8 +/- 0.7 mg/kg of caffeine (approx. 300 mg) for recreationally-trained males between the ages of 18 and 35 years. It remains unknown as to why the single-tower and two-tower reaction times were not significantly different between groups, as they included both audio and visual cues. It is hypothesized that the three-tower, multi-directional reaction time tests required the greatest movement and consequently the greatest task difficulty and overall mental and physical demand; thus, fatigue played a larger role in reaction time compared to the single- and two -tower tests. Independent of fatigue, caffeine could have influenced the multi-directional reaction tests because of the increased task complexity. Therefore, the improvements in the three-tower, multi-directional reaction time protocol after supplementation could have been attributed to both a reduction in fatigue as well as enhanced cognitive function. More research is required to determine the influence of the SUP used in the current investigation on reaction times using audio versus visual cues and various sport-specific movements.

An investigation by Fukuda et al. [15] examined whether a pre-workout supplement with similar ingredients (caffeine, creatine, amino acids) to the SUP used in the present study would impact anaerobic running capacity and critical velocity. Results showed no significant different between the SUP and the PL for critical velocity. However, it was reported that the SUP significantly increased anaerobic running capacity compared to the PL. In the present study, no significant differences were seen in anaerobic running capacity or intermittent critical velocity with the ingestion of the pre-workout supplement. However, the study by Fukuda et al. [15] was conducted in college-aged men and women and indicated an overall increase in anaerobic running capacity of 10.8% compared to the placebo with an increase in time to exhaustion at 110%, 105%, and 100% of peak velocity. Suggesting, the multi-ingredient supplement used in the investigation had effects on anaerobic performance only, which is similar to the current findings. Differences between findings could be related to the subjects and protocols used in the investigations. More research is needed regarding continuous anaerobic running capacity, critical velocity, and pre-workout supplements containing similar ingredients to those found in the SUP.

The SUP treatment decreased fatigue, increased energy, and improved choice reaction time (CRM15). These data suggest that at time point three (T3), around 95 min into the exercise protocol, as the subjects began to fatigue, the SUP caused a delay in fatigue and an increase in energy as evidenced by significantly faster choice reaction times (CRM15). Therefore, it appears that a single scoop (2 doses) of the SUP taken 20 min prior to exercise or training could allow for more total work with a higher quality (faster reactions) of work. Additionally, across all choice reaction time tests, mean values were faster for the SUP compared to the PL during time points two, three, and four with the exception of CRL15 at time point four, where the means were the same. At time point one; 20 min after ingestion, before any other exercises were completed, the SUP produced faster reactions in only four of the eight reaction tests. These findings suggest that the SUP began to effect performance only after the first round of testing, suggesting the largest factor related to improved performance was the ability of the SUP to delay fatigue and maintain high levels of energy.

Some limitations of this investigation were the use of lower percentages of speed at VO_2max to measure iCV and iARC compared to other investigations [14], which could have reduced the ability to find significant changes in anaerobic running performance, although it is more likely that a single dose of the SUP had little or no effect on iCV or iARC because creatine and beta alanine both require loading or prolonged use to see performance benefits, particularly in iCV or iARC.

Also, the fact that the SUP had multiple ingredients makes it impossible to identify which specific ingredients contributed to improved performance. Beta-alanine and creatine, two of the active ingredients in the pre-workout supplement (MusclePharm Assault™) have both been shown to improve anaerobic capacity [8, 21]. Most studies that investigated the effects of beta-alanine on exercise performance reported an increase in muscle carnosine [9, 21, 22]; although, those studies incorporated beta-alanine loading, stating that a longer supplementation period (≥ 4 weeks) could produce greater increases in muscle carnosine. In theory, a significant increase in muscle carnosine could result in an increase in muscle buffering capacity [22], translating to improvements in anaerobic running capacities by decreasing the accumulation of hydrogen ions. In the present study, no significant differences were seen in anaerobic running capacity and intermittent critical velocity with the one-time ingestion of the pre-workout supplement. Creatine and beta-alanine both require loading periods of several weeks to positively influence exercise performance. As a result, the only active ingredients likely to influence performance in this investigation were caffeine and B-vitamins.

Study limitations also relate to the characteristics of the subjects who participated in the current investigation. While the men were recreationally-trained with strength values in the 75^th and 90^th percentile (ACSM guidelines) for bench and leg press 1RM and VO_2max values in the 60^th percentile, the SUP may not have the same effects in men who are more anaerobically- or aerobically-trained. Nonetheless, the subjects in the current study should represent a large population of active men who regularly exercise. In addition, two-day food logs indicated subjects consumed an average of 31.5 +/- 109.4 mg of caffeine per day in their normal diet, and active men who regularly consume larger quantities of caffeine may not experience the same results as the men in the current investigation.

The overall findings of this investigation indicate that the supplement Assault™ can significantly improve perceived feelings of energy, focus, and alertness leading to a significant improvement in multi-directional choice reaction time and single-step choice reaction time with an auditory stimulus. Results also support that the SUP may reduce both feelings of fatigue and actual fatigue, which can result in performance increases in both muscular endurance and reaction time. Additionally, from a practical perspective, college-age males who recreationally participate in sports where auditory stimuli are abundant, such as team sports where verbal cues are encouraged may experience increases in performance after ingesting the pre-workout supplement used in the current study (Assault™).