Exercise training induces multiple adaptations within skeletal muscle that may improve local O2 delivery‐utilization matching (i.e., microvascular O2 pressure; PO2mv). We tested the hypothesis that increased nitric oxide (NO) bioavailability partly underlies improved muscle PO2mv kinetics from rest to contractions after exercise training. Rats were assigned to sedentary (S; n=18) or treadmill exercise trained (ET; n=10; 60 min/d, 5 d/wk, ~8 wks, 35 m/min; ‐14% grade) groups. PO2mv was measured via phosphorescence quenching in the spinotrapezius muscle at rest and during 1 Hz twitch contractions under control (CON, Krebs‐ Henseleit), SNP (NO donor; 300 μM) and L‐NAME (NO synthase blockade; 1.5 mM) superfusion. ET rats had greater VO2peak than S rats (S: 72 ± 2; ET: 81 ± 1 ml/kg/min; p<0.05). The rate of PO2mv fall during contractions was defined as ΔPO2mv/τ (where τ = time constant) and expressed in mmHg/s. Exercise training slowed ΔPO2mv/τ during CON (S: 1.46 ± 0.14; ET: 0.96 ± 0.14; p<0.05). Compared to CON, SNP slowed ΔPO2mv/τ only in S rats (S: 0.34 ± 0.06; ET: 0.65 ± 0.17; p<0.05) whereas L‐NAME speeded ΔPO2mv/τ only in ET rats (S: −12%; ET: 34%; p<0.05). Exercise training leads to greater microvascular oxygenation at the onset of contractions partly via increased NO bioavailability which likely constitutes an important mechanism for training‐induced metabolic adaptations.(AHA Midwest Affiliate, NIH HL‐108328)