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Skeletal muscle injury is a frequent event among athletes. The diagnosis using the classic blood markers sometimes produces unsatisfactory results due to the great interindividual variability. Therefore, the identification of reliable and acute biomarkers is important. This study verified the serum, salivary and urinary activity of biochemical markers indicative of musculoskeletal injury in response to acute muscle damage induced by intense physical exercise. A cross-sectional study was carried out with 27 football players with a mean age of 22.5 years. The athletes were submitted to a questionnaire before performing the physical activity and then evaluation of clinical parameters. Before the intense physical training (T0), 60 minutes (T1) and 24 hours (T2) after intense physical training, blood samples were collected by venipuncture that were conditioned in tubes with anticoagulant (heparin) and without anticoagulant and subsequently determined the activity of the enzymes Creatin Kinase (CPK), Adenosine deaminase (ADA), aldolase, Lactate dehydrogenase (LDH), Transaminase Glutamic Oxalacetic (TGO), Transaminase Glutamic Pyruvic (TGP) in serum, saliva and urine samples. Substances Reactive to Thiobarbituric Acid (TBARS). A significant increase in aldolase activity, ADA and CPK in serum, aldolase in saliva and ADA, CPK, TGO and TGP in urine was observed. There was also a significant increase in salivary and urinary TBARS after intense physical exercise. Together, the results showed that serum and urinary ADA activity, urinary and salivary TBARS, aldolase salivary and urinary TGO and TGP (p<0.01) could be effective and promising biomarkers of acute muscle injury, allowing a rapid assessment of physical exercise-induced lesions intense and muscular fatigue, contributing to the monitoring of athletes and thus to avoid more serious injuries and distance of the athletes. [ABSTRACT FROM AUTHOR]