Summary Objectives This study was designed to investigate the changes of circulating cell-free DNA (cfDNA) in boxer and rats undertook chronic high load and overtraining as well as its possible mechanisms after establishing a sensitive and simple method for detecting circulating cfDNA. Equipment and methods. Thirteen male boxers undertook high load training including 3-week high intensity and high volume trainings with a 4-week interval, while female rats completed high load training or overtraining, with 10 rats each group, in addition to 10 sedentary rats. Blood samples of all the participants were collected prior to the onset of training and at 36 h post the last session of training. The cfDNA level was detected by our established Alu-real-time PCR. Indicators monitoring high load training and overtraining including testosterone, cortisol (corticosterone in rat), Hb, creatine kinase (CK) and blood urea nitrogen (BUN), and reflecting oxidative stress such as glutathione peroxidase (GSH-Px) and peroxide index malonaldehyde (MDA), as well as exercise performance were determined. Results (1) Our real-time PCR targeting Alu or Alu homogenous gene was confirmed to detect circulating cfDNA simply and sensitively; (2) cfDNA levels were increased in high load training and overtraining rats by about 2.0- and 4.5-fold respectively, with a greater increase in overtraining rats. (3) Surged cfDNA levels were also observed by almost four times in high load training boxers, with no difference between high intensity and high volume trainings. Conclusions To our best knowledge, this study firstly suggested that the circulating cfDNA was likely to be a potential biomarker for chronic training and overtraining in human and rat, and the chronic training or overtraining-induced surge of circulating cfDNA might be originated from oxidative stress and muscle damage. Besides, a simple and sensitive quantitative PCR targeting Alu in human or Alu homologous gene in rat was suggested to detect circulating cfDNA concentration.