Dehydration (water loss >2.0% of body weight) has significant negative effects on physical and mental performance. In two studies the effects of minor hypo-hydration (water loss <1.0% of body weight) on CNS function, mood and cardiovascular functioning were measured. Study 1: On two mornings twelve male participants were exposed to a temperature of 30 °C for four hours and either did or did not drink two 150 ml glasses of water during that time. Study 2: Fifty-six (25 M) individuals were exposed to the same 30 °C environment and randomly allocated to either drink (2 × 150 ml) or not drink. When not given water 0.59% (Study 1) and 0.55% (Study 2) bodyweight was lost. Participant's heart rate variability (HRV) was measured, and they rated their thirst and mood. In study 1, participants participated in an fMRI protocol during which they completed a modified version of the Paced Auditory Serial Addition Test (PASAT), at the end of which they rated its difficulty. Decreases in fMRI BOLD activity in the orbito-frontal cortex, ventral cingulate gyrus, dorsal cingulate cortex, hypothalamus, amygdala, right striatum, post-central gyrus and superior parietal cortex were observed when participants were hypo-hydrated. These deactivations were associated with reduced HRV, greater perceived effort, and more anxiety. In study 2 declines in HRV were found to mediate the effect of hypo-hydration on ratings of anxiety. These data are discussed in relation to a model that describes how autonomic regulatory and interoceptive processes may contribute to the affective consequences of minor hypo-hydration.