Lactose intolerance is a major concern driving the growth of lactose-free foods including lactose-free infant formula. It is unknown what the metabolic consequence is of consumption of a formula where lactose has been replaced with corn syrup solids (CSS). Here, a randomized double-blinded intervention study was conducted where exclusively formula-fed infants were fed formula containing either lactose or CSS-based infant formula and compared with an equal number of exclusively breast-fed infants. Plasma metabolites and insulin were measured at baseline, 15, 30, 60, 90 and 120 min after feeding. Differences in plasma metabolite profiles for formula-fed infants included a rapid increase in circulating amino acids, creatinine and urea compared with breast-fed infants. At 120 min post-feeding, insulin was significantly elevated in formula-fed compared with breast-fed infants. Infants fed lactose-based formula had the highest levels of glucose at 120 min, and leucine, isoleucine, valine and proline at 90 and 120 min, whereas infants fed CSS-based formula had the lowest levels of non-esterified fatty acids at all time points, and glucose at 120 min. Overall, these differences highlight that changes in infant formula composition impact infant metabolism, and show that metabolomics is a powerful tool to help with development of improved infant formulas.