Dehydration can affect brain structure which has important implications for human health. In this study, we measured regional changes in brain structure following acute dehydration. Healthy volunteers received a structural MRI scan before and after an intensive 90‐min thermal‐exercise dehydration protocol. We used two techniques to determine changes in brain structure: a manual point counting technique using MEASURE, and a fully automated voxelwise analysis using SIENA. After the exercise regime, participants lost (2.2% ± 0.5%) of their body mass. Using SIENA, we detected expansion of the ventricular system with the largest change occurring in the left lateral ventricle (P = 0.001 corrected for multiple comparisons) but no change in total brain volume (P = 0.13). Using manual point counting, we could not detect any change in ventricular or brain volume, but there was a significant correlation between loss in body mass and third ventricular volume increase (r = 0.79, P = 0.03). These results show ventricular expansion occurs following acute dehydration, and suggest that automated longitudinal voxelwise analysis methods such as SIENA are more sensitive to regional changes in brain volume over time compared with a manual point counting technique. Hum Brain Mapp 2009. © 2007 Wiley‐Liss, Inc.