Background: Nutritional biomarkers are essential for assessing the impact of dietary interventions on health. Amino Acids (AA) serve as potential biomarkers due to their role in metabolic pathways, although the influence of combining macronutrients on AA metabolism remains unclear. Objective: This study aims to evaluate the effects of combining macronutrients (fat, protein, and fiber) on AA metabolism. Methods: A dietary intervention was conducted with 41 participants who were assigned to two groups: the Protein Fat (PF) group and the Protein Fat and Fiber (PFF) group. AA concentration was measured using LC-MS/MS. Results: Our findings indicated that combining macronutrients reduced plasma levels of AA with statistically significant differences between the two diets (p < 0.05 for EAA and BCAA; p < 0,01 for NEAA and AAA). Metabolic profile analysis showed differences between the groups, especially at 55 days (55 D) (R2X: 0.749; R2Y: 0.589; Q2: 0.582, p < 0.001) and 98 days (98 D) (R2X: 0.886; R2Y: 0.7; Q2: 0.605, p < 0.001). Five metabolites (leucine, asparagine, alanine, cysteine, and creatine) were the most influential factors in group differentiation (VIP score), with values ranging between 1.04 and 1.75. Conclusions: The combination of macronutrients led to a significant reduction in plasma amino acids and amines in the PFF group, with recovery post-intervention. These findings highlight the possibility that these metabolites are related to different health statuses.