Little is known about the metabolic differences between endurance and strength athletes in comparison with sedentary subjects under controlled conditions and about variation of the metabolome throughout one year. We hypothesized that (1) the resting metabolic profile differs between sedentary subjects and athletes and between perennially endurance- and strength-trained athletes and (2) varies throughout one year of training. We performed quantitative, targeted metabolomics (Biocrates MxP® Quant 500, Biocrates Life Sciences AG, Innsbruck, Austria) in plasma samples at rest in three groups of male adults, 12 strength-trained (weightlifters, 20 ± 3 years), 10 endurance-trained athletes (runners, 24 ± 3 years), and 12 sedentary subjects (25 ± 4 years) at the end of three training phases (regeneration, preparation, and competition) within one training year. Performance and anthropometric data showed significant (p < 0.05) differences between the groups. Metabolomic analysis revealed different resting metabolic profiles between the groups with acetylcarnitines, di- and triacylglycerols, and glycerophospho- and sphingolipids, as well as several amino acids as the most robust metabolites. Furthermore, we observed changes in free carnitine and 3-methylhistidine in strength-trained athletes throughout the training year. Regular endurance or strength training induces changes in the concentration of several metabolites associated with adaptations of the mitochondrial energy and glycolytic metabolism with concomitant changes in amino acid metabolism and cell signaling.