Animal studies have demonstrated an important role of peripheral mechanisms as contributors to exercise-induced hypoalgesia (EIH). Whether these same mechanisms contribute to EIH in humans is not known. In the current study, pain thresholds were assessed in healthy volunteers ( n = 36) before and after 5 min of high-intensity leg cycling exercise and an equivalent period of quiet rest. Pressure pain thresholds (PPTs) were assessed over the rectus femoris muscle of one leg and first dorsal interosseous muscles (FDIs) of both arms. Blood flow to one arm was occluded by a cuff throughout the 5-min period of exercise (or rest) and postexercise (or rest) assessments. Ratings of pain intensity and pain unpleasantness during occlusion were also measured. Pain ratings during occlusion increased over time (range, 1.5 to 3.5/10, all d > 0.63, P < 0.001) similarly in the rest and exercise conditions ( d < 0.35, P > 0.4). PPTs at all sites were unchanged following rest (range, −1.3% to +0.9%, all d < 0.05, P > 0.51). Consistent with EIH, exercise significantly increased PPT at the leg (+29%, d = 0.69, P < 0.001) and the nonoccluded (+23%, d = 0.56, P < 0.001) and occluded (+8%, d = 0.19, P = 0.003) unexercised arms. However, the increase in the occluded arm was significantly smaller ( d = −1.03, P < 0.001). These findings show that blocking blood flow to a limb during exercise attenuates EIH, suggesting that peripheral factors contribute to EIH in healthy adults. NEW & NOTEWORTHY This is the first demonstration in humans that a factor carried by the circulation and acting at the periphery is important for exercise-induced hypoalgesia. Further understanding of this mechanism may provide new insight to pain relief with exercise as well as potential interactions between analgesic medications and exercise.