Laser Doppler perfusion monitoring (LDPM) is a noninvasive technique for monitoring skin microcirculation. The aim of this article was to investigate the influence of fiber separation on clinical LDPM measurements. A dual-channel LDPM system was used in combination with a probe that consists of two sets of detection fibers, at 0.2 and 1.0 mm from the illuminating fiber. Measurements were performed at the big toe of 8 healthy subjects and 11 subjects who had vascular disorders. In most cases, fluxes detected at both fiber distances showed very similar fluctuations. For each fiber separation, flux values of healthy subjects and patients were not significantly different. Furthermore, skin temperature (range: 22-34 degrees C) influenced the toe's pulp microcirculation markedly, increasing similarly at both probe separations, with a higher flux at a separation of 1.0 mm than at 0.2 mm. The flux ratio signal, obtained by dividing the flux at 0.2 mm by the flux at 1.0 mm, was significantly different between the two groups (p &< 0.05). In conclusion, the flux detected in vivo by means of LDPM, is influenced by the distance between the optical fibers. Use of the flux ratio with a multiseparation probe deserves attention as it is a possible marker for discriminating normal tissue perfusion from pathological skin tissue perfusion, independently from tissue temperature.