It is known that the relevance of a peripheral stenosis for muscle function increases with exercise. Our intention was to investigate the impact of a moderate cuff stenosis (CS) at 120 mmHg of the superficial femoral artery on high-energy phosphate (HEP) metabolism during isotonic, incremental calf exercise. Serial phosphorus 31 magnetic resonance spectroscopy (31P MRS) and velocity-encoded phase-contrast MR imaging (VEPC MRI) were carried out in each leg of ten healthy male volunteers. Each leg underwent four increments of calf exercise (2, 3, 4 and 5 W) followed by recovery during separate exercise sessions with and without a CS at 120 mmHg. The serial 31P MRS measurements had a time resolution of 10 s. VEPC MRI was performed at the end of each increment during separate sessions. During all increments, we detected significant differences (P < 0.05) in the phosphocreatine (PCr) time constants and the amount of PCr hydrolysis between the sessions without and with CS. Regarding the time courses of the PCr, inorganic phosphate (Pi) and pH level, we observed significant differences (P < 0.002) during exercise and recovery. During both conditions, the end-increment PCr levels as well as blood flow correlated significantly with the mechanical power. The PCr time constants during exercise significantly correlated with the intramuscular pH, but not with blood flow or mechanical power. However, the PCr recovery time constants correlated significantly with blood flow and end-exercise pH. Our study shows that reduction of blood flow due to a peripheral stenosis results in a prolongation of PCr time constants, decreased PCr and pH level as well as increased Pi level during exercise. We believe that 31P MRS during incremental exercise might provide additional information for assessing the relevance of a peripheral stenosis and its impact on muscle function.