Skeletal muscle injury is a frequent event and diagnosis using the classical blood markers sometimes produces unsatisfactory results. Therefore, objective of the study was to detect new biomarkers in plasma, saliva and urine in response to acute muscle damage induced by physical exercise. A cross-sectional study was conducted with 27 American football players. Before the physical exercises (T0), 60 minutes (T1) and 24 hours (T2) after physical exercise, was determined the clinical, biochemical and molecular parameters, including ADA, TBARS, leukocytes, lymphocytes and comet assay. The serum ADA was significantly higher in T1 and T2, in the urine there was a significant increase in T1, in the saliva there was no significant differences. There was an increase in serum TBARS in T2, saliva and urine in T1. The leukocytes increased in T1 and decreased in T2. Through the comet assay was observed significant DNA damage in T1 and T2. Serum and urinary ADA activity, serum, urinary and salivary TBARS are robust and promising biomarkers of acute muscle injury and that the comet assay allows a quick and effective evaluation of DNA lesions induced by physical exercise and could be used to monitor athletes avoiding injuries that are more serious.