The analysis of blood constituents allows the detection of various physiological or pathological states when their values are increased or decreased in relation to a well-defined reference group or to themselves if monitored longitudinally. In the latter case, it is important to know the reference change value (RCV) or critical difference, which defines the percentage change that should be exceeded-given the analytical and biological variations inherent to a particular test, in that there is a significant difference between the two consecutive measurements. Our objective was to calculate the biological variation, analytical variation and RCV of the biochemical and hematological parameters in subjects undergoing 4 months of regular aerobic training. Blood samples (10 mL) were collected monthly from 56 male subjects (17-19 years old). Creatine kinase and aspartate aminotransferase activities, total cholesterol, triglycerides, uric acid, C-reactive protein, alpha-1-acid glycoprotein, creatinine and urea concentrations were measured in sera using an Autolab Boehringer analyzer. Hemogram were obtained from total blood using KX-21 N SYSMEX equipment. The RCV values for leukocytes and all biochemical analytes were elevated compared to the literature values of sedentary subjects. On the other hand, the RCV values for red blood cell count were slightly lower in physically active than in sedentary individuals. Knowledge of analyte RCV values within physically active subjects should improve the sensitivity/specificity of the hematological and biochemical alterations induced by training or the use of recombinant form of erythropoietin through blood parameter analysis, particularly in cases of longitudinal monitoring.