Although diagnostic CSF removal in patients with suspected normal pressure hydrocephalus (NPH) is performed frequently, its impact on changes of the global brain volume and volume of the ventricles has not been studied in detail. We examined 20 patients with clinical and radiological signs of NPH. These received MRI prior to and immediately after diagnostic CSF removal, either via lumbar puncture (TAP, n = 10) or via external lumbar drainage (ELD, n = 10). Changes in global brain volume were assessed using SIENA, a tool from the FSL software library. Additionally, we determined the change of the lateral ventricles' volume by manual segmentation. Furthermore, we recorded systematic clinical assessments of the key features of NPH. The median volume of CSF removed was 35 ml in TAP patients and 406 ml in ELD patients. Changes in global brain volume were found in both patient groups. Brain volume change was significantly larger in ELD patients than in TAP patients (p = 0.022), and correlated with the volume of CSF removal (r = 0.628, p = 0.004). Brain volume expansion was most pronounced adjacent to the lateral ventricles, but also detectable in the temporal and frontal regions. The median ventricular volume decreased after CSF removal. Ventricular volume reduction was more pronounced in ELD patients than in TAP patients. This study quantifies for the first time immediate volumetric changes of global brain tissue and of ventricles after diagnostic CSF removal in NPH patients. In particular, we report evidence that CSF removal results in a change of the brain volume rather than only a change of the brain's shape.