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Abstract A significant increase in circulating cell‐free DNA (cfDNA) occurs with physical exercise, which depends on the type of exertion and the duration. The aims of this study were as follows: (1) to investigate the time course of cfDNA and conventional markers of muscle damage from immediately after to 96 h after muscle‐damaging exercise; and (2) to investigate the relationship between cfDNA and indicators of primary (low‐frequency fatigue and maximal voluntary isometric contraction) and secondary (creatine kinase and delayed‐onset muscle soreness) muscle damage in young healthy males. Fourteen participants (age, 22 ± 2 years; weight, 84.4 ± 11.2 kg; height, 184.0 ± 7.4 cm) performed 50 intermittent drop jumps at 20 s intervals. We measured cfDNA and creatine kinase concentrations, maximal voluntary isometric contraction torque, low‐frequency fatigue and delayed‐onset muscle soreness before and at several time points up to 96 h after exercise. Plasma cfDNA levels increased from immediately postexercise until 72 h postexercise (P