Microvascular rarefaction (loss of functional capillaries) often parallels skeletal muscle dysfunction, but the relative contribution to performance decline is unknown because concurrent reductions in, e.g. muscle fibre size and oxidative capacity occur in pathologies such as chronic heart failure. To investigate the effect of microvascular rarefaction on muscle performance in otherwise healthy tissue, capillary perfusion was reduced in the rat extensor digitorum longus (EDL) by arteriolar blockade using microsphere injections. Bilateral EDL twitch force and fatigue-resistance were determined by stimulating at 10Hz to elicit isometric contractions for 180s. Carotid blood pressure and bilateral femoral artery blood flow were monitored simultaneously. Microspheres were injected following an initial bout of stimulation (to establish baseline conditions) via the superficial epigastric artery (a branch of the femoral artery). To assess capacity for adaptive remodelling during chronic ischaemia, functional overload of EDL was performed by extirpation of a muscle synergist coupled with injections of microspheres, followed by 2-week recovery period. Histological assessment of capillaries was enabled by injection of fluorescent Dextran and subsequent lectin staining. The ratio of perfused: unperfused capillaries could then be calculated. Fatigue index (maximum force at end /beginning of stimulation) in control EDL was 46.96 ± 8.75%, and decreased in proportion to microsphere injection (n = 15; r2 0.566; P < 0.001). A decrease in exercise hyperaemia was also observed after microsphere injection (r2 = 0.255; P = 0.006). Contralateral EDL had unchanged fatigue resistance (r2 0.022; P = 0.467) and vascular conductance (r2 = 0.001; P = 0.891). Impaired muscle performance was correlated with a reduction in perfused capillaries (r2 0.462, P = 0.031). Interestingly, chronically reduced capillary perfusion did not influence adaptive remodelling of EDL and mechanical performance did not differ from control (n = 14; P = 0.990), despite attenuated exercise hyperaemia (P < 0.05). These experimental data highlight the sensitivity of muscle endurance to acute changes in microvascular perfusion. Conversely, muscle function is not deleteriously affected by arteriolar blockade in the long-term, possibly as a result of shear stress insensitive angiogenesis. By quantifying the interaction between performance and perfusion, we aim to provide therapeutic targets for skeletal muscle dysfunction in patients with chronic heart failure.