This study investigated the effects of high intensity interval training (HIIT) and β-hydroxy-β-methylbutyric free acid (HMBFA) on the onset of neuromuscular fatigue in healthy young men and women. Thirty-seven subjects (22 men and 15 women; mean ± SD age = 22.8 ± 3.4yrs) completed an incremental cycle ergometer test (GXT) to exhaustion. During the GXT, electromyography (EMG) signals from the right vastus lateralis were recorded to determine the power output at the onset of neuromuscular fatigue (PWCFT), and peak wattage was used to assign individual training loads. After baseline testing (PRE), subjects were randomly assigned to one of three groups: control (C, n = 9), training with placebo (P, n = 14), or training with supplementation (S, n = 14). Subjects assigned to P and S completed 12 HIIT sessions over 4 weeks while subjects assigned to C were asked to maintain their normal diet and activity patterns. After 4 weeks, subjects returned for post-testing (POST). The PWCFT values (W) were determined using a DMAX method. The EMG amplitude root mean square (µVrms) values were used to generate a third-order polynomial regression (3PR) representing the increase in µVrms versus time of the GXT. The onset of fatigue (TF) was defined as the x-value (time, s) of the point on the 3PR that measured the maximal perpendicular distance from the line between the first and last data points. TF was used to estimate PWCFT according to the equation: PWCFT = PO + a · (n/N), where PO is the power output of the stage in which TF occurred, a is the increment in power output between GXT stages (25W), n is the difference (s) between TF and the beginning of the stage during which TF occurred, and N is the duration of a stage (120s). A two-way repeated measures ANOVA was used to identify group × time interaction for PWCFT. If a significant interaction occurred, one-way factorial ANOVAs were used. Fisher's least significant difference post hoc comparisons were performed between groups. If a significant main effect occurred, dependent samples t-tests with Bonferroni corrections (p = [0.05/3] = 0.017) were performed across time for each group. The two-way ANOVA resulted in a significant interaction (F = 6.69, p = 0.004). Follow-up analysis with one-way ANOVA resulted in no difference among groups at PRE (F = 0.87, p = 0.43), however a significant difference was shown for POST values (F = 5.46, p = 0.009). Post-hoc analysis among POST values showed significant differences between S and both P (p = 0.034) and C (p = 0.003). No differences (p = 0.226) were noted between P and C. Paired samples t-tests detected significant changes following HIIT for S (p < 0.001) and P (p = 0.016), but no change in C (p = 0.473). Results of this study indicate that HIIT was effective in delaying the onset of fatigue, but supplementation with HMBFA in conjunction with HIIT was more effective than HIIT alone. An increase in PWCFT represents an increase in the maximal power output an individual can sustain without eliciting fatigue. Therefore, HIIT can be used to improve performance in both endurance activities as well as intermittent sports. In addition, HMBFA supplementation is a simple method that can be used to maximize the benefits of HIIT. Acknowledgments: Metabolic Technologies, Inc. provided the supplement and funding for this study.