This study determined the beverage hydration index (BHI) and postprandial cardiac autonomic activity after consuming an isotonic beverage (IB) compared to distilled water (DW). Twenty-two participants (50% female; mean ± SD; age, 27 ± 3 year; height, 169.1 ± 12.6 cm; weight, 73.3 ± 13.8 kg; BF%, 23 ± 10%) completed two experimental trials where they consumed 1 L DW or an IB; after which urine volume and cardiac autonomic activity was measured through 240 min. Cardiac autonomic activity was quantified using heart rate (HR), log transformed heart rate variability measures (root mean square of successive R–R intervals; RMSSD; low frequency, LF; and high frequency, HF) and systolic time intervals (pre-ejection period, PEP). BHI was significantly greater after IB consumption at min 0 (MD [95% CI]; 1.31 [0.35, 2.27]), 180 min (0.09 [0.022, 0.16]), and 240 min (0.1 [0.03, 0.17]) compared to DW (p = 0.031). Net fluid balance was significantly greater in IB than DW at 180 min (90 [−16.80, 196.81]) and 240 min (106 [−13.88, 225.88]) (p = 0.037). HR decreased over time in both beverage trials but was higher following IB ingestion at 0 min (3.9 [−2.42, 10.22]), 30 min (5.3 [−0.94, 11.54]), and 60 min (2.7 [−3.42, 8.82]) (p = 0.0002). lnHF was greater 30 min post DW ingestion compared to IB (0.45 [−0.23, 1.13]) (p = 0.039). IB promotes greater fluid retention capacity compared to DW within 4 hours of consumption. The variations in cardiac autonomic measures may warrant further investigation in clinical populations (i.e., patients with autonomic failure).