In a model of chronic hind limb ischaemia, we examined whether impaired muscle blood flow, particularly during exercise, is partly due to modification of the reactivity of skeletal muscle resistance vessels by prolonged low blood flow. Two or 5 weeks after unilateral iliac artery ligation, terminal (A4) and preterminal (A3) arterioles of extensor digitorum longus muscle were viewed by intravital microscopy using epi-illumination, and diameter changes to topical application of endothelium-dependent (bradykinin, acetylcholine) and endothelium-independent (adenosine, sodium nitroprusside and noradrenaline) agonists measured. Chronic ischaemia had no effect on resting diameters of A3 or A4 vessels. Two weeks after ligation, dilation to bradykinin was attenuated by 75% for A3 and 50% for A4 arterioles (p < 0.01 vs. control) and responses to acetylcholine were reversed from dilation to constriction (A3: control diameter change +29%, 2-week-ligated –17%; A4: control 18%, 2-week-ligated –13%). Five weeks after ligation, these effects were still apparent and, additionally, dilation to adenosine and sodium nitroprusside and constriction to noradrenaline were reduced. Thus, impaired dilation, most likely due to endothelial dysfunction, is an early manifestation of altered reactivity in the microcirculation of chronically ischaemic muscles, with functional impairment of vascular smooth muscle as a later consequence. These changes occurred despite modest improvements in muscle blood flow and perfusion pressure over the same time. These changes will act to the detriment of blood flow in contracting muscles and could limit the outcome of interventions to restore flow such as angioplasty or surgical bypass.