Body mass changes during ultra-endurance performances have been described for running, cycling and for swimming in a heated pool. The present field study of 20 male and 11 female open-water swimmers investigated the changes in body composition and hydration status during an ultra-endurance event. Body mass, both estimated fat mass and skeletal muscle mass, haematocrit, plasma sodium concentration ([Na+]) and urine specific gravity were determined. Energy intake, energy expenditure and fluid intake were estimated. Males experienced significant reductions in body mass (-0.5 %) and skeletal muscle mass (-1.1 %) (P < 0.05) during the race compared to females who showed no significant changes with regard to these variables (P > 0.05). Changes in percent body fat, fat mass, and fat-free mass were heterogeneous and did not reach statistical significance (P > 0.05) between gender groups. Fluid intake relative to plasma volume was higher in females than in males during the ultra-endurance event. Compared to males, females' average increase in haematocrit was 3.3 percentage points (pp) higher, urine specific gravity decrease 0.1 pp smaller, and plasma [Na+] 1.3 pp higher. The observed patterns of fluid intake, changes in plasma volume, urine specific gravity, and plasma [Na+] suggest that, particularly in females, a combination of fluid shift from blood vessels to interstitial tissue, facilitated by skeletal muscle damage, as well as exercise-associated hyponatremia had occurred. To summarise, changes in body composition and hydration status are different in male compared to female open-water ultra-endurance swimmers.