Short, middle, and long-term exercise, as well as the relative intensity of the physical effort, may influence a broad array of laboratory results, and it is thereby of pivotal importance to appropriately differentiate the 'physiologic' from the 'pathological' effects of exercise. Therefore, the values of some biomarkers in physically active subjects may be cautiously interpreted since the results may fall outside the conventional reference ranges. It has been demonstrated that middle and long-term endurance and/or strenuous exercise triggers transient elevations of muscular and cardiac biomarkers. However, no data have been published about the effect of short-term maximal exercise test on the most useful muscular, hepatic and cardiovascular biomarkers. The aim of the present study was to assess the baseline concentrations of muscular, hepatic, and cardiovascular makers between trained and untrained subjects, along with changes induced by maximal exercise test. We measured C reactive protein (CRP), procalcitonin (PCT), gamma glutamyltransferase (GGT), creatine kinase-MB isoenzyme (CK-MB), Hs-TnT, NT-proBNP, CK, LDH, AST, and ALT in serum samples of physically active (trained) and physically inactive (sedentary) male collected before, immediately after a maximal exercise test and after a 30-min recovery period. Trained subjects tend to have significantly raised base concentrations of CK, CK-MB, ALT, and LDH compared to sedentary individuals, and this can be clearly interpreted as a mild injury of skeletal muscle. A single maximal exercise was also effective to transiently increase the concentrations of NT-proBNP, but not those of Hs-TnT, thus suggesting that the cardiac involvement is mostly benign in nature.