Using near-infrared spectroscopy (NIRS) and the tracer indocyanine green (ICG), we quantified blood flow in calf muscle and around the Achilles tendon during plantar flexion (1-9 W). For comparison, blood flow in calf muscle was determined by dye dilution in combination with magnetic resonance imaging measures of muscle volume, and, for the peritendon region, blood flow was measured by (133)Xe washout. From rest to a peak load of 9 W, NIRS-ICG blood flow in calf muscle increased from 2.4+/-0.2 to 74+/-5 ml x 100 ml tissue(-1) x min(-1), similar to that measured by reverse dye (77+/-6 ml x 100 ml tissue(-1) x min(-1)). Achilles peritendon blood flow measured by NIRS-ICG rose with exercise from 2.2+/-0.5 to 15.1+/-0.2 ml x 100 ml(-1) x min(-1), which was similar to that determined by (133)Xe washout (2.0+/-0.6 to 14.6+/-0.3 ml x 100 ml tissue(-1) x min(-1)). This is the first study using NIRS and ICG to quantify regional tissue blood flow during exercise in humans. Due to its high spatial and temporal resolution, the technique may be useful for determining regional blood flow distribution and regulation during exercise in humans.