Neurostimulation/neurorecording are tools to study, diagnose, and treat neurologic/psychiatric conditions. Both techniques depend on volume conduction between scalp and excitable brain tissue. We examine how neurostimulation with TMS is affected by hydration status, a physiologic variable which can influence the volume of fluid spaces/cells, excitability and cellular/global brain functioning. Compared to dehydration, we expected rehydration to show signs of macroscopic and microscopic volume changes including decreased scalp-cortex distance (brain closer to stimulator) and astrocyte swelling-induced glutamate release. Normal healthy adult participants (32, 9 male) had common motor TMS measures taken in a repeated measures design from dehydrated (12-hour overnight fast/thirst) and rehydrated (identical dehydration protocol followed by rehydration with 1 L water in 1 hour) testing days. The target region was left primary motor cortex hand area. Response at the target muscle was recorded with electromyography. Urinalysis confirmed hydration status. Motor hotspot shifted in half of participants. Motor threshold decreased in rehydration, indicating increased excitability. Even after re-dosing/re-localizing TMS to the new threshold/hotspot, rehydration still showed evidence of increased excitability: recruitment curve measures generally shifted upwards and SICF was increased. SICI, LICI, LICF, and CSP were relatively unaffected. The hydration perturbations were mild/subclinical, based on the magnitude/speed and urinalysis. Motor TMS measures showed evidence of expected physiologic changes of osmotic challenges. Hydration may be a source of variability affecting techniques dependant on brain volumes/volume conduction. These concepts are important for researchers/clinicians using such techniques or dealing with the wide variety of disease processes involving water balance.