Purpose We tested the hypothesis that incremental ramp cycling exercise performed in the supine position ( S ) would be associated with an increased reliance on muscle deoxygenation (deoxyheme) in the deep and superficial vastus lateralis (VLd and VLs, respectively) and the superficial rectus femoris (RFs) when compared to the upright position ( U ). Methods 11 healthy men completed ramp incremental exercise tests in S and U . Pulmonary V ˙ O 2 was measured breath-by-breath; deoxyheme was determined via time-resolved near-infrared spectroscopy in the VLd, VLs and RFs. Results Supine exercise increased the overall change in deoxyheme from baseline to maximal exercise in the VLs ( S : 38 ± 23 vs. U : 26 ± 15 μM, P  < 0.001) and RFs ( S : 36 ± 21 vs. U : 25 ± 15 μM, P  < 0.001), but not in the VLd ( S : 32 ± 23 vs. U : 29 ± 26 μM, P  > 0.05). Conclusions The present study supports that the impaired balance between O 2 delivery and O 2 utilization observed during supine exercise is a regional phenomenon within superficial muscles. Thus, deep muscle defended its O 2 delivery/utilization balance against the supine-induced reductions in perfusion pressure. The differential responses of these muscle regions may be explained by a regional heterogeneity of vascular and metabolic control properties, perhaps related to fiber type composition.