The purpose of this study was to use traditional physical assessments combined with a metabolomic approach to compare the anthropometric, physical fitness level, and serum fasting metabolic profile among U22 soccer players at different competitive levels. In the experimental design, two teams of male U22 soccer were evaluated (non-elite = 20 athletes, competing in a regional division; elite = 16 athletes, competing in the first division of the national U22 youth league). Earlobe blood samples were collected, and metabolites were extracted after overnight fasting (12 h). Untargeted metabolomics through Liquid Chromatograph Mass Spectrometry (LC-MS) analysis and anthropometric evaluation were performed. Critical velocity was applied to determine aerobic (CV) and anaerobic (ARC) capacity. Height (non-elite = 174.4 ± 7.0 cm; elite = 176.5 ± 7.0 cm), body mass index (non-elite = 22.1 ± 2.4 kg/m2; elite = 21.9 ± 2.3 kg/m2), body mass (non-elite = 67.1 ± 8.8 kg; elite = 68.5 ± 10.1 kg), lean body mass (non-elite = 59.3 ± 7.1 kg; elite = 61.1 ± 7.9 kg), body fat (non-elite = 7.8 ± 2.4 kg; elite = 7.3 ± 2.4 kg), body fat percentage (non-elite = 11.4 ± 2.4%; elite = 10.5 ± 1.7%), hematocrit (non-elite = 50.2 ± 4.0%; elite = 51.0 ± 4.0%), CV (non-elite = 3.1 ± 0.4 m/s; elite = 3.0 ± 0.2 m/s), and ARC (non-elite = 129.6 ± 55.7 m; elite = 161.5 ± 61.0 m) showed no significant differences between the elite and non-elite teams, while the multivariate Partial Least Squares Discriminant Analysis (PLS-DA) model revealed a separation between the elite and non-elite athletes. Nineteen metabolites with importance for projection (VIP) >1.0 were annotated as belonging to the glycerolipid, sterol lipid, fatty acyl, flavonoid, and glycerophospholipid classes. Metabolites with a high relative abundance in the elite group were related in the literature to a better level of aerobic power, greater efficiency in the recovery process, and improvement of mood, immunity, decision making, and accuracy, in addition to acting in mitochondrial preservation and electron transport chain maintenance. In conclusion, although classical physical assessments were not able to distinguish the teams at different competitive levels, the metabolomics approach successfully indicated differences between the fasting metabolic profiles of elite and non-elite teams.