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Serial T1WI and ¹H-MR spectroscopy data were acquired in 15 healthy individuals at normohydration, at 12 hours of dehydration, and during 1 hour of oral rehydration. Osmotic challenges were monitored by serum measures, including osmolality and hematocrit. With dehydration, serum osmolality increased by 0.67% and brain tissue fluid decreased by 1.63%. MR imaging morphometry demonstrated corresponding decreases of cortical thickness and volumes of the whole brain, cortex, white matter, and hypothalamus/thalamus. These changes reversed during rehydration. The authors conclude that it is essential to control for hydration levels in studies on brain morphometry and metabolism in order to avoid confounding the findings.