The study of nutrient timing has become an important and popular aspect of sports nutrition, exercise training, performance, and recovery 1 . The idea of nutrient timing was initiated by post-workout supplementation and has further spread to research on the timing of pre-exercise nutritional strategies 1 . Traditional nutritional interventions prior to training have focused on carbohydrate administration, while more current literature has supported a combination of amino acids, protein, creatine and caffeine as effective supplements for improving performance 2 3 4 5 6 . While the ergogenic effects from these individual ingredients are generally supported, the practical importance of product-specific research has become an area of increasing demand. Paradoxically, product-specific research often tests a blend of ingredients that provides a direct application of the research findings for consumers, but is unable to pinpoint the effects of individual ingredients. Furthermore, integrating nutritional supplements into research designs that use realistic exercise training protocols allows for impactful sport-specific practical applications. Since many team sports, such as football, basketball, hockey and soccer utilize repeated bouts of short sprints separated by active recovery periods, interval running may be applicable to many sports and sensitive to nutritional supplements that are designed to delay high-intensity, exercise-induced fatigue. In fact, evidence exists to support the use of high-intensity interval training (HIIT) strategies to improve performance 7 , however, only a few studies have examined HIIT combined with nutritional supplementation 8 9 10 11 12 13 .

The physiological demand of HIIT elicits rapid metabolic and cardiovascular adaptations, including increased exercise performance, muscle buffering capacity, aerobic capacity (VO₂peak) and fat oxidation 8 14 15 16 17 . Furthermore, HIIT results in diminished stores of adenosine tri-phosphate (ATP), phosphocreatine (PCr) and glycogenic substrates as well as the accumulation of metabolites adenosine di-phosphate (ADP), inorganic phosphate (P_i), and hydrogen ions (H⁺) 18 . Therefore, HIIT may cause several physiological adaptations within a relatively brief training period, making it a practical time-efficient tool to examine training- and supplement-induced changes in performance. Although the work to rest ratio of HIIT protocols vary, the current study and others utilizing a 2:1 work:rest strategy have been effective for improving VO₂max, time to exhaustion 9 11 19 , muscle buffering capacity, and lactate threshold 8 . Additionally, the same HIIT strategy that is used in the present study has been employed to evaluate the effects of creatine 9 10 , beta-alanine 11 , and sodium bicarbonate 8 supplementation on measures of performance. Therefore, it is possible that the training outcomes measured after a period of HIIT may be sensitive to nutritional supplements that are designed to prolong the acute factors associated with fatigue. More so, the active ingredients in the current pre-workout supplement have potential to improve performance. Caffeine or caffeine containing supplements acting as a central nervous system stimulant 20 have been suggested to augment catecholamine concentrations promoting fat utilization sparing intramuscular glycogen resulting in an improvement in performance 21 22 . PCr, a major component of biological buffering has been reported to be significantly increased with Cr supplementation 23 24 . Increasing total Cr stores can result in greater pre-exercise PCr availability, improved muscle buffer capacity and an acceleration of PCr resynthesis during recovery 25 26 . Additionally, branched chain amino acids (BCAA's; leucine, isoleucine, and valine) are suggested to be the primary amino acids oxidized during intense exercise 27 . When supplementing with BCAAs prior to exercise, research suggests an improvement in protein synthesis, reduction in protein degradation, ultimately improving recovery 27 28 29 .

Interval training is generally used to elicit both anaerobic and aerobic training adaptations due to the large physiological spectrum of demands 30 . The critical power test (CP), originally proposed by Monod and Scherrer 31 , characterizes both anaerobic work capacity (AWC) and aerobic parameters (CP). The CP test has been shown to be reliable in measuring aerobic and anaerobic parameters as well as changes with high-intensity training 10 32 33 34 . Hughson et al. 35 applied the concept of CP to running, which characterized the term critical velocity (CV) as the running-based analogue of CP. Thus, CV is defined as the maximal running velocity that can be maintained for an extended period of time using only aerobic energy stores. In contrast, the anaerobic running capacity (ARC) is the distance that can be run at a maximal velocity using only anaerobic energy sources. As described by Housh et al. 36 , the CV test involves a series of runs to exhaustion at various supramaximal running velocities to determine the relationship between time to exhaustion and velocity. The hyperbolic relationship between velocity and time to exhaustion can then be used to calculate total distance (total distance = velocity × time). Plotting total distance as a function of time for each velocity results in a mathematical model to quantify CV (slope of the line) and ARC (y-intercept), which defines the indirect method of evaluating both aerobic and anaerobic capabilities, respectively 35 37 .

Recent evidence has shown that interval training with two-minute work bouts, similar to the HIIT in the present study, exerts a significant influence on aerobic abilities (CV), rather than the anaerobic improvements (AWC) demonstrated by the CP test 32 38 . Training at intensities of 100% and 105% of VO₂peak on a cycle ergometer elicited a 15% 32 and 13% 38 increase in aerobic capacity, respectively. Training at higher intensities for shorter durations (i.e. 60 sec) may be more advantageous for anaerobic improvements 33 , although this hypothesis has not been evaluated using the CV test. Likewise, the efficacy of single-ingredient supplements has been assessed using the CP model. For example, creatine supplementation has been shown to improve AWC, which is primarily limited by the amount of energy available from stored ATP and phosphocreatine (PCr) 39 . However, less conclusive evidence is available on the effects of creatine on CP 10 40 41 . It is possible that combining the use of a multi-ingredient, pre-workout supplement with HIIT may further delineate the anaerobic and aerobic demands of training as measured by CV and ARC using the running-based CV test. Therefore, the purpose of the present study was to examine the effects of a pre-workout supplement combined with three weeks of HIIT on aerobic and anaerobic running performance, training volume, and body composition. To date, no one has examined the combined effects of high-intensity interval running with a pre-workout nutritional supplement.

Table 3 contains the means and standard errors for each of the dependent variables (CV, ARC, VO₂max, %BF, FM, and LBM). In addition, there were no significant differences (p > 0.05) between the GT and PL groups at the pre-training testing session.

The results of the present study indicated that acute ingestion of the current pre-exercise drink (GT) containing a combination of cordyceps sinensis, caffeine, creatine (Kre-Alkalyn^®), whey protein, branched chain amino acids, arginine AKG, citrulline AKG, rhodiola, and vitamin B6 and B12 may improve running performance over a 3-week training period. When combined with HIIT, GT ingestion improved CV, VO₂max, lean body mass, and total training volume when compared to the PL and HIIT group. In addition, although not significant, the fact that LBM changes were positive for the GT group and negative for the PL group (Figure 2-E) suggests that GT may aid in maintaining LBM during the course of HIIT for three weeks.

While this may be the first study to examine a pre-workout supplement in combination with HIIT, previous research has examined the efficacy of similar, separate ingredients on exercise training and performance. However, since most previous studies examine blended supplements that often include various ingredients and dose combinations, it is difficult to directly compare many previous studies. One primary ingredient in the GT supplement, caffeine, has been used as an effective ergogenic aid by acting as a stimulant, reducing feelings of fatigue, and increasing times to exhaustion 22 45 46 47 . Caffeine has been shown to primarily influence longer-duration endurance exercise by 20-50% 48 and resting metabolic rate 45 49 50 51 . The benefits of caffeine supplementation for higher-intensity exercise, similar to those in the current study (90%-115% VO₂max), are less conclusive 52 53 . For example, assessing anaerobic power using a Wingate test after a range of caffeine doses (3.2-7 mg/lb) resulted in no improvements 52 53 while Anselme et al. demonstrated a 7% increase in anaerobic power after 6 mg/kg of caffeine consumption 54 . In addition, a recent report by Wiles et al. demonstrated improvements in performance during a bout of short-duration, high-intensity cycling and mean power output following 5 mg/kg of caffeine 55 . The results of the present study indicated that the pre-exercise GT drink improved aerobic performance (CV) and training volume, but did not alter the ARC. It is possible that the caffeine in GT may be partly responsible for the increases in CV and training volume. However, the independent effects of caffeine cannot be directly assessed in the present study.

Previous studies have suggested that the ergogenic effects of caffeine may be proportional to the amount of caffeine administered 56 57 58 . Most studies have utilized 3-9 mg/kg of caffeine when demonstrating improvements in performance 48 , while one study showed that as little 2 mg/kg increased cycling performance 58 . Yet another study demonstrated that 201 mg of caffeine was not sufficient for increasing run time to exhaustion 59 . In the present study, the pre-exercise GT supplement contained only 100 mg of caffeine in one serving. Since the range of body mass values for the participants in the present study was 46.1 kg to 108.9 kg, the relative caffeine doses were 1.0 - 2.2 mg/kg, which is lower than the previously suggested ergogenic doses. Therefore, although caffeine may have contributed to improvements in aerobic performance and training volume in the present study, it is possible that there were synergistic effects from other GT ingredients.

One concern about the ergogenic doses of caffeine is that relatively high levels of urinary caffeine concentrations are banned by both the National Collegiate Athletics Association (NCAA) and the International Olympic Committee (IOC). The NCAA and IOC limits for urinary caffeine concentrations are 15 μg/ml and 12 μg/ml, respectively. In a well-controlled study 60 the average urinary concentration of caffeine was 14 μg/ml after the ingestion of 9 mg/kg. In an earlier study, Pasman et al. (1995) demonstrated that 9 and 13 mg/kg of caffeine consumption resulted in urinary caffeine concentrations that exceeded the International Olympic Committee's (IOC's) limit of 12 μg/ml in some subjects. However, 5 mg/kg of caffeine did not exceed or even approach 12 μg/ml in any subject 61 . Since the relative caffeine dose range for the GT supplement in the present study was 1.0 - 2.2 mg/kg for an absolute dose of 100 mg of caffeine per serving, it is highly unlikely that the caffeine in GT would cause urinary concentrations anywhere near the limits set forth by the NCAA or IOC. Therefore, although not tested specifically in this study, the GT supplement may be safe for consumption by NCAA and IOC athletes as it pertains to caffeine concentrations.

A large amount of literature exists demonstrating that short-term high-dose (20 g/day for 5-7 days) creatine supplementation is effective for increasing total muscle phosphocreatine stores 23 24 and improving maximal intermittent exercise 23 25 62 63 64 and lean body mass 64 65 66 67 68 . However, the data on short-term low-dose creatine supplementation is less supported, with a minimum of 3 g/day for at least 28 days necessary to elicit increases in muscle creatine stores 69 . The current pre-workout GT drink provided 1.5 g/day of creatine on testing and training days only for a total of 15 days, which was below the minimum recommended dose. A similar study by Thompson and colleagues used a comparable combination of training (swimming) and 2 g of creatine daily for six weeks and demonstrated no effects of the creatine supplementation or training on muscle creatine concentration, anaerobic performance, or aerobic indices 70 . Thus, although the creatine content of the GT supplement may not fully explain the improvements in CV and training volume, the combination with the other GT ingredients may have been influential for intermittent recovery between sprint bouts as well as helping to maintain LBM.

The BCAAs in GT may have also played a role in improving CV and training volume as well as maintaining LBM. BCAAs may be the primary amino acids oxidized during intense exercise 27 and have been suggested as fundamental for protein synthesis 27 28 29 . Studies have demonstrated that the ingestion of BCAA supplements prior to exercise has augmented protein synthesis and reduced protein degradation, which may ultimately enhance recovery time 27 29 . Furthermore, BCAAs may conceivably enhance performance in all-out running, similar to the current study by improving mental focus allowing participants to run harder and longer 71 72 . Again, however, the GT supplement contained approximately 1 g of BCAAs which is lower than other effective dosing protocols (7.5-12 g). There was also approximately 9 g of whey protein concentrate in the GT supplement. Although whey protein has not been directly shown to improve running performance when consumed a priori, the fact that whey protein also contains relatively high concentrations of the BCAAs may indirectly suggest that the BCAAs in combination with whey protein may influence performance by enhancing recovery between training bouts and maintaining LBM 73 74 75 76 .

Cordyceps sinensis (or simply cordyceps) is commonly used in traditional Chinese medicine, and it is derived from a fungus that grows on several species of caterpillars at relatively high altitudes 77 . It has been suggested that cordyceps may be an anti-oxidant during intense exercise 78 and may also improve VO₂max 79 . In two reviews by Zhu et al. 77 80 , it was suggested that cordyceps sinensis may act through the autonomic nervous system to improve respiration, blood flow, and tissue oxygenation. One study has demonstrated improvements in VO₂max in sedentary men 79 with relatively high doses (4.5 g/d for 6 weeks) of cordyceps. However, with lower doses (2.5 g) similar to what is found in GT in the present study, there were no ergogenic effects of cordyceps reported in previous studies on VO₂max 81 82 83 in healthy, active men. Thus, given the conflicting evidence, cordyceps may be another ingredient in GT that acted synergistically to improve CV and training volume in the present study.

The role that the remaining ingredients in the GT supplement (ex. Citrulline and rhodiola) may play is not completely evident. Citrulline is a non-essential amino acid that may increase lactate absorption, enhance ATP resynthesis, and delay fatigue during intense exercise 84 85 . While evidence is limited in humans, citrulline may have influenced ATP/PCr resynthesis during HIIT bouts thereby enhancing the training volume. Furthermore, rhodiola may act as a stimulant to optimize serotonin and dopamine levels 86 . Acute supplementation (i.e., 2 days) has been shown to increase time to exhaustion and VO₂peak by acting as an antioxidant and reducing the perception of fatigue 87 88 89 90 . Together these ingredients may have positively influenced CV and training volume, however, this speculation cannot be proven in the current study.

In conclusion, the results of this study indicate that the acute ingestion of the pre-exercise GT supplement containing 100 mg of caffeine, 1.5 g creatine, 1 g BCAAs, 9 g whey protein, 2.5 g of cordyceps sinensis and a combined 0.75 g of citrulline and rhodiola, taken prior to HIIT for three weeks can significantly improve CV and total training volume when compared to HIIT and PL. Furthermore, the maintenance of and trend toward an improvement in LBM suggests that GT may be helpful in maintaining lean mass during intense training periods. Although there was not a single ingredient in GT that could solely account for the improvements, it is likely that the combination of relatively low doses of several ingredients (caffeine, creatine, BCAAs, whey protein, and cordyceps) may be responsible for the increases in aerobic performance, training volume, and the maintenance of lean mass.

The authors declare that they have no competing interests.

AES was the primary author of the manuscript and played an important role in study design, data collection and assessment. DHF and KLK played an important role in data collection and manuscript preparation. JRS was the senior author and played an important role in the grant procurement, study design, data analysis and manuscript preparation. All authors have read and approved the final manuscript.