Functional vasodilation in arterioles is impaired with chronic ischemia. We sought to examine the impact of chronic ischemia and age on skeletal muscle resistance artery function. To examine the impact of chronic ischemia, the femoral artery was resected from young (2-3mo) and adult (6-7mo) mice and the profunda femoris artery diameter was measured at rest and following gracilis muscle contraction 14 days later using intravital microscopy. Functional vasodilation was significantly impaired in ischemic mice (14.4±4.6% vs. 137.8±14.3%, p<0.0001 n=8) and non-ischemic adult mice (103.0±9.4% vs. 137.8±14.3%, p=0.05 n=10). In order to analyze the cellular mechanisms of the impairment, a protocol was developed to apply pharmacological agents to the experimental preparation while maintaining tissue homeostasis. Endothelial and smooth muscle dependent vasodilation were impaired with ischemia, 39.6 ± 13.6% vs. 80.5 ± 11.4% and 43.0 ± 11.7% vs. 85.1 ± 10.5%, respectively. From this data, it can be supported that smooth muscle dysfunction is the reason for the observed impairment in arterial vasodilation.