International Journal of Exercise Science 10(4): 550-559, 2017. Betaine (BT) supplementation improves selected markers of physical performance, however, the mechanism(s) by which this change occurs remains largely unknown. Some speculate that BT may increase circulating nitrate concentrations, improving physical performance by augmentation of endothelial nitric oxide production. The purpose of this study was to investigate the effects of acute BT supplementation and exercise on plasma nitrate levels and related cardiovascular response (CVR). Placebo and BT trials were administered in a cross-over, randomized, double-blind, and counterbalanced fashion. Ten healthy college-aged volunteers consumed either a 250 ml placebo (carbohydrate-electrolyte beverage, CHO) or 250 ml CHO + 2.5 g BT. Subjects rested for 45 min, then cycled for 30 min at 60 rpm with a resistance of 2.5% body weight. Blood was drawn before and 45 min after BT supplementation, and immediately post exercise to assess plasma nitrate levels. Repeated measures ANOVA across treatments and times assessed differences in plasma nitrate and CVR variables with an alpha level set at 0.05. No significant interactions nor differences between groups were found for plasma nitrate levels or CVR variables with acute BT supplementation. A significant time effect (p \u3c 0.013) for all CVR variables was found and expected due to the effect of exercise. Acute BT supplementation did not increase plasma nitrate levels nor alter CVR at rest or during light to moderate cycling