The purpose of this study was to examine the effect of venous occlusion per se on forearm muscle blood flow, as determined by the near-infrared spectroscopy (NIRS) venous occlusion technique (NIRS-VOT). NIRS data were obtained from the flexor digitorum superficialis (FDS) muscle on the dominant arm of 16 young, ostensibly healthy participants (14 men and two women; 30 ± 6 year; 73 ± 7 kg). Participants completed a series of five venous occlusion trials while seated at rest, and a series of 12 venous occlusion trials during a reactive hyperaemia induced by 5 min of forearm arterial occlusion. The NIRS-VOT was used to assess FDS muscle blood flow ((Formula presented.)), beat-by-beat, over the first four cardiac beats during venous occlusions. (Formula presented.) was also reported as a cumulative value, wherein the first two, first three and first four cardiac beats were used to calculate muscle blood flow. We observed that (Formula presented.) was highest when calculated over the first cardiac beat during venous occlusions performed at rest and throughout reactive hyperaemia (P<0˙05). Moreover, the inclusion of more than one cardiac beat in the calculation of (Formula presented.) underestimated muscle blood flows, irrespective of the prevailing level of arterial inflow. These findings support the idea that venous occlusion per se affects the measurement of (Formula presented.) via the NIRS-VOT. Accordingly, it is recommended that (Formula presented.) is determined over the first cardiac beat when using the NIRS-VOT to assess microvascular blood flow of human forearm muscles.