Peripheral arterial disease (PAD) is a systemic atherosclerotic vascular disease characterized by impaired skeletal muscle perfusion and mitochondrial respiration. Over time, persistent exposure to muscle ischemia eventually leads to muscle atrophy, myopathy, and mitochondrial dysfunction in PAD patients. In this study we investigated the utilization of diffusion tensor imaging to characterize the pathophysiological changes of calf muscles before and after graded plantar flexion exercise. At rest, the clinically worse leg of the PAD subjects had higher diffusivity in the calf muscles. Significant greater increases of diffusivity in the post-exercise muscle of PADs than the HCs suggest acute exercise-related muscle damage.