Physical effort can elicit differential adaptive changes in the tissues of trained versus untrained rats. Proteolytic activity in the extracellular matrix could be engaged in such adaptation due to its influence on the elasticity of tissues. The effects were investigated of a single physical effort on the activity of elastase, cathepsin K, and plasmin in the skeletal muscles, heart muscles, and aortas of untrained (UT, n=30) and trained (T, n=30) rats. T rats underwent 6 weeks of endurance training. After the last training session, T and UT rats were divided randomly into 3 subgroups. Ten rats from each group (Tpre, n=10) and (UTpre, n=10) were sacrificed. The other 20 rats from each group performed 60 min. of aerobic exercise and were sacrificed immediately post exercise (T0h, n=10; UT0h, n=10) or 3h later (T3h, n=10; UT3h, n=10). Enzyme activity was measured fluorometrically. Cathepsin K and plasmin activity increased in the soleus muscles of UT0h versus UTpre, plasmin activity increased also in UT3h versus UTpre. Elastase, cathepsin K and plasmin activity increased in the heart muscles of T0h and T3h versus Tpre. No aortic differences were observed. Thus, a single bout of physical effort elicited different responses in tissues of T versus UT rats. Increased proteolytic enzyme levels in muscles could influence tissue remodeling. Unchanged aortic cathepsin K levels may help prevent aortic remodeling and neointima formation.