Ex vivo skeletal muscle arteriole endothelial function is impaired in claudicating patients with PAD, and this is associated with attenuated skeletal muscle mitochondrial respiration. In vivo skeletal muscle oxygen delivery and utilization capacity are compromised in PAD, and this may be due to microcirculatory and mitochondrial dysfunction. These results suggest that targeting skeletal muscle arteriole function may lead to improvements in skeletal muscle mitochondrial respiration, oxygen delivery, and utilization capacity in claudicating patients with PAD. , Peripheral artery disease (PAD) is an atherosclerotic disease that impairs blood flow and muscle function in the lower limbs. A skeletal muscle myopathy characterized by mitochondrial dysfunction and oxidative damage is present in PAD; however, the underlying mechanisms are not well established. We investigated the impact of chronic ischemia on skeletal muscle microcirculatory function and its association with leg skeletal muscle mitochondrial function and oxygen delivery and utilization capacity in PAD. Gastrocnemius samples and arterioles were harvested from patients with PAD ( n = 10) and age-matched controls (Con, n = 11). Endothelium-dependent and independent vasodilation was assessed in response to flow (30 μL·min −1 ), acetylcholine, and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiration was quantified by high-resolution respirometry, microvascular oxygen delivery, and utilization capacity (tissue oxygenation index, TOI) were assessed by near-infrared spectroscopy. Vasodilation was attenuated in PAD ( P textless 0.05) in response to acetylcholine (Con: 71.1 ± 11.1%, PAD: 45.7 ± 18.1%) and flow (Con: 46.6 ± 20.1%, PAD: 29.3 ± 10.5%) but not SNP ( P = 0.30). Complex I + II state 3 respiration ( P textless 0.01) and TOI recovery rate were impaired in PAD ( P textless 0.05). Both flow and acetylcholine-mediated vasodilation were positively associated with complex I + II state 3 respiration ( r = 0.5 and r = 0.5, respectively, P textless 0.05). Flow-mediated vasodilation and complex I + II state 3 respiration were positively associated with TOI recovery rate ( r = 0.8 and r = 0.7, respectively, P textless 0.05). These findings suggest that chronic ischemia attenuates skeletal muscle arteriole endothelial function, which may be a key mediator for mitochondrial and microcirculatory dysfunction in the PAD leg skeletal muscle. Targeting microvascular dysfunction may be an effective strategy to prevent and/or reverse disease progression in PAD. NEW & NOTEWORTHY Ex vivo skeletal muscle arteriole endothelial function is impaired in claudicating patients with PAD, and this is associated with attenuated skeletal muscle mitochondrial respiration. In vivo skeletal muscle oxygen delivery and utilization capacity is compromised in PAD, and this may be due to microcirculatory and mitochondrial dysfunction. These results suggest that targeting skeletal muscle arteriole function may lead to improvements in skeletal muscle mitochondrial respiration and oxygen delivery and utilization capacity in claudicating patients with PAD.