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The aim of this study was to quantify changes in brain volume after exercise/heat-induced hypohydration in man.Eight active men completed intermittent exercise in a warm environment, until 2.9% ± 0.1% of body mass was lost. Subjects remained hypohydrated for 2 h after the end of exercise. Brain volume was measured before, immediately after, and 1 and 2 h after exercise using magnetic resonance imaging (Philips 3T Achieva, AE Eindhoven, The Netherlands). Measures of subjective feelings and core body temperature were also monitored. Blood samples were drawn to determine serum electrolyte concentrations and osmolality and to allow calculation of changes in blood and plasma volumes.Brain volume was not influenced by hypohydration (0.2% ± 0.4%; effect size (ES) = 0.2, P = 0.310). Reductions in ventricular (4.0% ± 1.8%; ES = 4.6, P < 0.001) and cerebrospinal fluid (3.1% ± 1.9%; ES = 3.3, P = 0.003) volumes were observed after exercise. Compared with preexercise levels, serum osmolality was elevated throughout the 2-h postexercise period (+10 ± 2 mOsm·kg−1, P < 0.001). Core temperature increased from 37.1°C ± 0.3°C at rest to 39.3°C ± 0.5°C at the end of exercise (P = 0.001).These data demonstrate that brain volume remains unchanged in response to moderate hypohydration and presence of serum hyperosmolality, suggesting that mechanisms are in place to defend brain volume.