Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. Accordingly, we hypothesized this concept could be applied to the field of exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of non-treated human participants using next generation sequencing (NGS) and to clearly define the dynamics regarding the amounts of spcfDNA-fragments upon extreme exercise, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from non-treated participants. We confirmed the TaqMan-qPCR assay had a high sensitivity and found the spcfDNA sequence abundance was 16,600-fold higher than a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of the levels of spcfDNA-fragments upon running a full marathon. Quantities of the spcfDNA fragments were significantly increased post marathon. Furthermore, the amounts of spcfDNA fragments strongly correlated with the numbers of white blood cells and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive response markers to extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology on the human.