The purpose of this study was to examine the acute endothelial, cardiovascular, and performance responses to L-arginine intake by assessing flow-mediated dilation (FMD) and various indicators (e.g., heart rate, heart rate variability (HRV), blood pressure, torque) both before and after resistance exercise. Thirty (15 male, 15 female) physically active participants (mean +/- SD: age 20.4 +/- 1.8 years, height 176.9 +/- 10.2 cm, body mass 76.0 +/- 12.2 kg) volunteered for a randomized, cross-over, double-blind, placebo-controlled clinical trial. Participants completed five sets of elbow extension-flexion exercise after consumption of either 3 g L-arginine or 3 g of placebo. There was a significant decline in post-exercise elbow extension (p = 0.014) and flexion peak torque (p < 0.001). FMD response after exercise was ~5.8% less than before resistance exercise (L-arginine and placebo data pooled, p < 0.001). Baseline brachial artery diameter significantly increased post-FMD (p < 0.001), post-resistance exercise (p < 0.001), and post-resistance exercise FMD (p < 0.001). There were significant time effects for HRV when expressed as the square root of the mean of the sum of squares of differences between adjacent RR intervals (RMSSD) or the proportion of differences between adjacent normal (NN) RR intervals that exceed 50 ms (pNN50) (all p-values < 0.05), but there were no treatment or interaction effects (all p-values > 0.05). We conclude the increased vasodilation due to acute resistance exercise was not enhanced by acute supplementation with L-arginine nor was exercise performance augmented. Further, the relative contribution of sympathetic nervous system input increased with resistance exercise but was not influenced by the addition of L-arginine.