Abstract Elevated plasma levels of cell-free mitochondrial DNA (cf-mDNA), a cell damage-associated molecular pattern (DAMP), contribute to neutrophil activation and inflammation in trauma patients, and may occur in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we conducted trial by time experiments for plasma cf-mDNA response to prolonged moderate aerobic exercise, and to eccentric exercise. Seven healthy moderately-trained young men (n=7) completed treadmill exercise trials for 90 min at 60% VO2 max, and resting control trials, and blood was sampled immediately prior to (Pre), during (at +18, +54, and +90 min), and after (R40) trials. A significant difference in cf-mDNA response was observed between exercise and control trials. Specifically, cf-mDNA levels differed at +54 and +90 (with or without plasma volume (PV) shift correction). A significant time effect was observed, with relative cf-mDNA levels declining at +54 and +90 during exercise. These results suggest increased clearance or reduced release of plasma cf-mDNA with exercise. Declines in cf-mDNA were also seen after eccentric exercise. Our finding of cf-mDNA decline with prolonged moderate treadmill and eccentric exercise contrasts with studies by others involving exhaustive short-term treadmill exercise, where cf-mDNA levels were unchanged, and highlights differences between exercise- and trauma-induced inflammation.