Voluntary hyperventilation (HV) provokes hemoconcentration due to a loss of fluid from the intravascular space. In 10 healthy male volunteers the hypothesis was tested whether HV increases transcapillary fluid shift into the interstitial compartment. For this purpose, fluorescent light intensity (FLI) alterations after intravenous injection of sodium fluorescein (Na fluorescein) before and during 3 min of HV were determined. Concomitantly, temperature and microvascular skin flux (laser Doppler fluxmetry, LDF) were recorded continuously. Hematocrit and serum proteins, as markers of hemoconcentration, increased significantly from 41.2 ± 2.3 to 42.7 ± 2.0% (p = 0.0023) and from 69.5 ± 3.4 to 72.9 ± 3.0 g/l (p = 0.0005, respectively). Skin temperature and LDF showed no changes during HV compared to baseline levels. Interstitial FLI indicating transcapillary diffusion of Na fluorescein was significantly higher (p < 0.001) during HV compared to the values recorded during the baseline period. The exact mechanism of enhanced transcapillary diffusion of Na fluorescein is not known. The distinct increase in FLI without a significant change in microvascular skin flux suggests an HV-induced increase in capillary pressure or an enhancement in capillary permeability for water and small solu