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Purpose Hamstring injuries in soccer reportedly increase towards the end of the matches’ halves as well as with increased match frequency in combination with short rest periods, possibly due to acute or residual fatigue. Therefore, this study aimed to investigate the effects of acute and residual muscle fatigue on exercise-induced hamstring muscle damage. Methods A three-armed randomized-controlled trial, including 24 resistance-trained males, was performed allocating subjects to either a training group with acute muscle fatigue + eccentric exercise (AF/ECC); residual muscle fatigue + eccentric exercise (RF/ECC) or a control group with only eccentric exercise (ECC). Muscle stiffness, thickness, contractility, peak torque, range of motion, pain perception, and creatine kinase were assessed as muscle damage markers pre, post, 1 h post, and on the consecutive three days. Results Significant group × time interactions were revealed for muscle thickness (p = 0.02) and muscle contractility parameters radial displacement (Dm) and contraction velocity (Vc) (both p = 0.01), with larger changes in the ECC group (partial η2 = 0.4). Peak torque dropped by an average of 22% in all groups; stiffness only changed in the RF/ECC group (p = 0.04). Muscle work during the damage protocol was lower for AF/ECC than for ECC and RF/ECC (p = 0.005). Conclusion Hamstring muscle damage was comparable between the three groups. However, the AF/ECC group resulted in the same amount of muscle damage while accumulating significantly less muscle work during the protocol of the damage exercise. Trial registration This study was preregistered in the international trial registration platform (WHO; registration number: DRKS00025243).

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