Significance Long-duration spaceflight induces detrimental changes in human physiology due to microgravity. One example is a cephalic fluid shift. Here, we prospectively investigated the quantitative changes in cerebrospinal fluid (CSF) volume of the brain ventricular regions in space crew by means of a region of interest, observer-independent analysis on structural brain MRI scans. MRI scans were collected before the mission, shortly after and 7 mo after return to Earth. We found a significant increase in lateral and third ventricles at postflight and a trend to normalization at follow-up, but still significantly increased ventricular volumes. The observed spatiotemporal pattern of CSF compartment enlargement and recovery points to a reduced CSF resorption in microgravity as the underlying cause.