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Exercise-induced hypoalgesia (EIH) is a decrease in the pain sensitivity after exercise. Individuals with chronic pain show less EIH after one exercise session compared with pain-free individuals possibly due to pain in exercising muscles. The primary aim of this randomized controlled cross-over study was to compare the EIH response at the exercising thigh muscle following exercises performed with painful vs. non-painful muscles. Secondary aims were to explore if a reduced EIH response was confined to the painful muscle, and whether the muscle pain intensity and the EIH responses were negatively associated.