The microvascular partial pressure of oxygen (Pmvo2) kinetics following the onset of exercise reflects the relationship between muscle O2delivery and uptake (V̇o2). Although AMP-activated protein kinase (AMPK) is known as a regulator of mitochondria and nitric oxide metabolism, it is unclear whether the dynamic balance of O2delivery and V̇o2at exercise onset is dependent on AMPK activation level. We used transgenic mice with muscle-specific AMPK dominant-negative (AMPK-DN) to investigate a role for skeletal muscle AMPK on Pmvo2kinetics following onset of muscle contractions. Phosphorescence quenching techniques were used to measure Pmvo2at rest and across the transition to twitch (1 Hz) and tetanic (100 Hz, 3–5 V, 4-ms pulse duration, stimulus duration of 100 ms every 1 s for 1 min) contractions in gastrocnemius muscles (each group n = 6) of AMPK-DN mice and wild-type littermates (WT) under isoflurane anesthesia with 100% inspired O2to avoid hypoxemia. Baseline Pmvo2before contractions was not different between groups ( P > 0.05). Both muscle contraction conditions exhibited a delay followed by an exponential decrease in Pmvo2. However, compared with WT, AMPK-DN demonstrated 1) prolongation of the time delay before Pmvo2began to decline (1 Hz: WT, 3.2 ± 0.5 s; AMPK-DN, 6.5 ± 0.4 s; 100 Hz: WT, 4.4 ± 1.0 s; AMPK-DN, 6.5 ± 1.4 s; P < 0.05), 2) a faster response time (i.e., time constant; 1 Hz: WT, 19.4 ± 3.9 s; AMPK-DN, 12.4 ± 2.6 s; 100 Hz: WT, 15.1 ± 2.2 s; AMPK-DN, 9.0 ± 1.7 s; P < 0.05). These findings are consistent with the presence of substantial mitochondrial and microvascular dysfunction in AMPK-DN mice, which likely slows O2consumption kinetics (i.e., oxidative phosphorylation response) and impairs the hyperemic response at the onset of contractions thereby sowing the seeds for exercise intolerance.