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Abstract Background It remains unclear if ad libitum water drinking, as a hydration strategy, prevents exercise-associated hyponatremia (EAH) during prolonged exercise. The aim of this study was to determine the incidence of EAH within the broader context of fluid regulation among soldiers performing a 40-km route-march ingesting water ad libitum. Methods Twenty-eight healthy male soldiers participated in this observational trial. Pre- and post-exercise body mass, blood and urine samples were collected. Blood samples were assessed for serum sodium ([Na+]), glucose, creatinine, urea nitrogen (BUN), plasma osmolality, creatine kinase (CK), and plasma arginine vasopressin (AVP) concentrations. Plasma volume (PV) was calculated using hematocrit and hemoglobin. Urine samples were analyzed for osmolality and [Na+]. Water intake was assessed by weighing bottles before, during and after the march. The mean relative humidity was 55.7% (21.9–94.3%) and the mean dry bulb temperature was 27.1 °C (19.5 °C - 37.0 °C) during the exercise. Results Twenty-five soldiers (72 ± 10 kg) (Mean ± SD) completed the march in 09:11 ± 00:43 (hr:min). Participants consumed 736 ± 259 ml/h of water and lost 2.8 ± 0.9 kg (4.0% ± 1.4%, P 1000 mOsmol/kg H2O) and plasma AVP. No single hydration strategy likely prevents EAH, but hypernatremia (cellular dehydration) was not seen despite > 2% body mass losses and high urine osmolality.