The present study evaluated, using a well-controlled dehydration protocol, the effects of 24 h fluid deprivation (FD) on selected mood and physiological parameters. In the present cross-over study, twenty healthy women (age 25 (SE 0.78) years) participated in two randomised sessions: FD-induced dehydration v. a fully hydrated control condition. In the FD period, the last water intake was between 18.00 and 19.00 hours and no beverages were allowed until 18.00 hours on the next day (23-24 h). Water intake was only permitted at fixed periods during the control condition. Physiological parameters in the urine, blood and saliva (osmolality) as well as mood and sensations (headache and thirst) were compared across the experimental conditions. Safety was monitored throughout the study. The FD protocol was effective as indicated by a significant reduction in urine output. No clinical abnormalities of biological parameters or vital signs were observed, although heart rate was increased by FD. Increased urine specific gravity, darker urine colour and increased thirst were early markers of dehydration. Interestingly, dehydration also induced a significant increase in saliva osmolality at the end of the 24 h FD period but plasma osmolality remained unchanged. The significant effects of FD on mood included decreased alertness and increased sleepiness, fatigue and confusion. The most consistent effects of mild dehydration on mood are on sleep/wake parameters. Urine specific gravity appears to be the best physiological measure of hydration status in subjects with a normal level of activity; saliva osmolality is another reliable and non-invasive method for assessing hydration status.