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 96h 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±2years; weight, 84.4±11.2kg; height, 184.0±7.4cm) performed 50 intermittent drop jumps at 20s 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 96h after exercise. Plasma cfDNA levels increased from immediately postexercise until 72h postexercise (P<0.01). Elevation of postexercise cfDNA was correlated with both more pronounced low-frequency fatigue (r=-0.52, P=3.4×10-11) and delayed-onset muscle soreness (r=0.32, P= 0.00019). Levels of cfDNA change in response to severe primary and secondary muscle damage after exercise. Levels of cfDNA exhibit a stronger correlation with variables related to primary muscle damage than to secondary muscle damage, suggesting that cfDNA is a more sensitive marker of acute loss of muscle function than of secondary inflammation or damaged muscle fibres.