Premenopausal women express reduced blood pressure and risk of cardiovascular disease relative to age-matched men. This purportedly relates to elevated estrogen levels increasing nitric oxide synthase (NOS) activity and NO-mediated vasorelaxation. We tested the hypotheses that female rat skeletal muscle would: 1 ) evince a higher O 2 delivery-to-utilization ratio (Q̇ o 2 /V̇ o 2 ) during contractions; and 2 ) express greater modulation of Q̇ o 2 /V̇ o 2 with changes to NO bioavailability compared with male rats. The spinotrapezius muscle of Sprague-Dawley rats (females = 8, males = 8) was surgically exposed and electrically-stimulated (180 s, 1 Hz, 6 V). OxyphorG4 was injected into the muscle and phosphorescence quenching employed to determine the temporal profile of interstitial P o 2 (P o 2is , determined by Q̇ o 2 /V̇ o 2 ). This was performed under three conditions: control (CON), 300 µM sodium nitroprusside (SNP; NO donor), and 1.5 mM N ω -nitro- l -arginine methyl ester ( l -NAME; NOS blockade) superfusion. No sex differences were found for the P o 2is kinetics parameters in CON or l -NAME ( P > 0.05), but females elicited a lower baseline following SNP (males 42 ± 3 vs. females 36 ± 2 mmHg, P < 0.05). Females had a lower ΔP o 2is during contractions following SNP (males 22 ± 3 vs. females 17 ± 2 mmHg, P < 0.05), but there were no sex differences for the temporal response to contractions ( P > 0.05). The total NO effect (SNP minus l -NAME) on P o 2is was not different between sexes. However, the spread across both conditions was shifted to a lower absolute range for females (reduced SNP baseline and greater reduction following l -NAME). These data support that females have a greater reliance on basal NO bioavailability and males have a greater responsiveness to exogenous NO and less responsiveness to reduced endogenous NO. NEW & NOTEWORTHY Interstitial P o 2 (P o 2is ; determined by O 2 delivery-to-utilization matching) plays an important role for O 2 flux into skeletal muscle. We show that both sexes regulate P o 2is at similar levels at rest and during skeletal muscle contractions. However, modulating NO bioavailability exposes sex differences in this regulation with females potentially having a greater reliance on basal NO bioavailability and males having a greater responsiveness to exogenous NO and less responsiveness to reduced endogenous NO.