Muscle deoxygenation (i.e., deoxyHb + Mb) during exercise assesses the matching of oxygen delivery (Q̇O2) to oxygen utilization (V̇O2). Until now limitations in near-infrared spectroscopy (NIRS) technology did not permit discrimination of deoxyHb + Mb between superficial and deep muscles. In humans, the deep quadriceps is more highly vascularized and oxidative than the superficial quadriceps. Using high-power time-resolved NIRS, we tested the hypothesis that deoxygenation of the deep quadriceps would be less than in superficial muscle during incremental cycling exercise in eight males. Pulmonary V̇O2 was measured and muscle deoxyHb + Mb was determined in the superficial vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF-s) and the deep rectus femoris (RF-d). deoxyHb + Mb in RF-d was significantly less than VL at 70% (67.2 ± 7.0 vs. 75.5 ± 10.7 μM) and 80% (71.4 ± 11.0 vs. 79.0 ± 15.4 μM) of peak work rate (WR(peak)), but greater than VL and VM at WR(peak) (87.7 ± 32.5 vs. 76.6 ± 17.5 and 75.1 ± 19.9 μM). RF-s was intermediate at WR(peak) (82.6 ± 18.7 μM). Total hemoglobin and myoglobin concentration and tissue oxygen saturation were significantly greater in RF-d than RF-s throughout exercise. The slope of deoxyHb + Mb increase (proportional to Q̇O2/V̇O2) in VL and VM slowed markedly above 70% WR(peak), whereas it became greater in RF-d. This divergent deoxygenation pattern may be due to a greater population of slow-twitch muscle fibers in the RF-d muscle and the differential recruitment profiles and vascular and metabolic control properties of specific fiber populations within superficial and deeper muscle regions.