The aim of our study was to investigate the effects of inspiratory negative pressure breathing (NPBin) via impedance threshold device on breathing pattern and body fluid distribution in cosmonauts during spaceflight. The lung function (volumes and flows, breath-hold time) was estimated in details before NPBin exposure as well. The 16 cosmonauts participated in study on ISS board: they were examined before, during spaceflight and after landing. All cosmonauts included in the study were well trained for providing the experimental procedure by themselves. The mask with impedance threshold (−20 cm H2O on inhale) was used. Breathing rate, intramask pressure and electrical impedance of head, body and limbs have been monitoring before (5 min) and during NPBin (20 min). The obtained data has shown no significant differences in lung volumes and flows in comparison of preflight and spaceflight values. Generally, NPBin led to significant decrease of breathing rate. No statistically significant changes in electrical impedance of body and head at NPBin compared with tidal breathing were found. The effectiveness of NPBin as a preventing means for fluid shifts in cosmonauts (astronauts) during spaceflight is discussed and needs the further studies.