Maintaining a proper fluid balance is important during exercise as athletes are prone to develop dehydration during exercise. Although several factors may regulate the fluid balance, little is known about the role of sex during prolonged moderate-intensity exercise. Therefore, we compared body mass changes and fluid balance parameters in men vs women in a large heterogeneous group of participants during prolonged exercise. Ninety-eight volunteers walked 30–50 km at a self-selected pace. Exercise duration (8 h, 32 min) and intensity (69% HRmax) were comparable between groups. Men demonstrated a significantly larger change in body mass than women (−1.6% vs −0.9%, respectively, P < 0.001) and a higher incidence of dehydration (defined as ≥2% body mass loss) compared with women (34% vs 12%, respectively, odds ratio = 4.2, 95% CI = 1.1–16.7). Changes in blood sodium levels were significantly different between men (+1.5 mmol/L) and women (−0.4 mmol/L), while 27% of the men vs 0% of the women showed postexercise hypernatremia (sodium levels ≥ 145 mmol/L). Moreover, men demonstrated a significantly lower fluid intake (2.9 mL/kg/h) and higher fluid loss (5.0 mL/kg/h) compared with women (3.7 and 4.8 mL/kg/h, respectively). Taken together, our data suggest that men and women demonstrate different changes in fluid balance in response to a similar bout of exercise.