Strength-endurance type of sport can lead to modification of human beings' physiological status. The present study aimed to investigate the alteration of metabolic phenotype or biochemical compositions in professional athletes induced by long-term training by means of a novel systematic tool, metabolomics. Resting venous blood samples of junior and senior male rowers were obtained before, after 1-week and 2-week training. Venous blood from healthy male volunteers as control was also sampled at rest. Endogenous metabolites in serum were profiled by GC/TOF-MS and multivariate statistical technique, i.e. principal component analysis (PCA) and partial least squares projection to latent structures & discriminant analysis (PLS-DA), were utilized to process the data. Significant metabolomic difference was observed between the professional athletes and control subjects. Long-term strength and endurance training induced distinct separation between athletes of different exercise seniority, and training stage-related trajectory of the two groups of athletes was clearly shown along with training time. However, most of these variations were not observed by commonly biochemical parameters, such as haemoglobin, testosterone and creatine kinase. The identified metabolites contributing to the classification included alanine, lactate, -D-Methylglucopyranoside, pyroglutamic acid, cysteine, glutamic acid, citric acid, free fatty acids, valine, glutamine, phenylalanine, tyrosine and so on, which were involved in glucose metabolism, oxidative stress, energy metabolism, lipid metabolism, amino acid metabolism. These findings suggest that metabolomics is a promising and potential tool to profile serum of professional athletes, make a deep insight into physiologic states and clarify the disorders induced by strength-endurance physical exercise.