BACKGROUND: The combination of maternal obesity in early pregnancy and high protein intake in infant formula feeding might predispose to obesity risk in later life. METHODS: This study assesses the impact of breast- or formula-feeding (differing in protein content by 1.65 or 2.7g/100 kcal) on the metabolism of term infants from overweight and obese mothers. From birth to 3 mo of age, infants received exclusively either breast- or starter formula-feeding and until 6 mo, exclusively either a formula designed for this study or breast-feeding. From 6 to 12 mo, infants received complementary weaning food. Metabonomics was conducted on the infants' urine and stool samples collected at the age of 3, 6, and 12 mo. RESULTS: Infant formula-feeding resulted in higher protein-derived short-chain fatty acids and amino acids in stools. Urine metabonomics revealed a relationship between bacterial processing of dietary proteins and host protein metabolism stimulated with increasing protein content in the formula. Moreover, formula-fed infants were metabolically different from breast-fed infants, at the level of lipid and energy metabolism (carnitines, ketone bodies, and Krebs cycle). CONCLUSION: Noninvasive urine and stool metabolic monitoring of responses to early nutrition provides relevant readouts to assess nutritional requirements for infants' growth.