Hydration practices may confound heart rate variability (HRV) measurements when collected in the pre-training period. We aimed to determine the effects of ingesting a hypertonic, sugar-sweetened sports beverage on HRV and hemodynamic parameters in physically active young men. Fifteen subjects consumed 591 mL of Gatorade (6% carbohydrate, approximately 330 mOsmol/kg), 591 mL water, or 10 mL water (control) in random order on separate days following overnight fasting. HRV and hemodynamics were evaluated in 5-min windows immediately before (T1) and 5-10 min (T2), 25-30 min (T3), 40-45 min (T4), and 55-60 min (T5) post-drinking. Root-mean square of successive differences and the standard deviation of normal RR intervals increased post-water intake at all time-points relative to T1 (P < 0.05). No increases were observed post-Gatorade intake, though small effect sizes were noted at T2 and T3 (P > 0.05, ES = 0.27-0.32). Systemic vascular resistance increased at T2 post-Gatorade intake and at T2 and T3 post-water intake (P < 0.05). No interactions were observed for blood pressure measures, stroke volume, or cardiac output. Gatorade does not evoke cardiovascular adjustments to the same magnitude as water. Practitioners should wait at least 45 min to record HRV post-Gatorade intake and >60 min post-water intake. Novelty: Equal volumes of cold water and Gatorade produce inequivalent cardiac-autonomic and hemodynamic responses. HRV responses of greater amplitude and duration were observed following intake of water versus Gatorade. Failure to account for recent fluid intake may result in misinterpretation of autonomic status.