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This study explored the effects of physical activity (PA) and ambient temperature on water turnover and hydration status. Five-hundred seventy three healthy men and women (aged 20-60 years) from Spain, Greece and Germany self-reported PA, registered all food and beverage intake, and collected 24-h urine during seven consecutive days. Fasting blood samples were collected at the onset and end of the study. Food moisture was assessed using nutritional software to account for all water intake which was subtracted from daily urine volume to allow calculation of non-renal water loss (i.e., mostly sweating). Hydration status was assessed by urine and blood osmolality. A negative association was seen between ambient temperature and PA (r = -0.277; p < 0.001). Lower PA with high temperatures did not prevent increased non-renal water losses (i.e., sweating) and elevated urine and blood osmolality (r = 0.218 to 0.163 all p < 0.001). When summer and winter data were combined PA was negatively associated with urine osmolality (r = -0.153; p = 0.001). Our data suggest that environmental heat acts to reduce voluntary PA but this is not sufficient to prevent moderate dehydration (increased osmolality). On the other hand, increased PA is associated with improved hydration status (i.e., lower urine and blood osmolality).