ABSTRACT Background The use of biomarkers of food intake (BFIs) in blood and urine has shown great promise for assessing dietary intake and complementing traditional dietary assessment tools whose use is prone to misreporting. Objective Untargeted LC-MS metabolomics was applied to identify candidate BFIs for assessing the intake of milk and cheese and to explore the metabolic response to the ingestion of these foods. Methods A randomized controlled crossover study was conducted in healthy adults [5 women, 6 men; age: 23.6 ± 5.0 y; BMI (kg/m2): 22.1 ± 1.7]. After a single isocaloric intake of milk (600 mL), cheese (100 g), or soy-based drink (600 mL), serum and urine samples were collected postprandially up to 6 h and after fasting after 24 h. Untargeted metabolomics was conducted using LC-MS. Discriminant metabolites were selected in serum by multivariate statistical analysis, and their mass distribution and postprandial kinetics were compared. Results Serum metabolites discriminant for cheese intake had a significantly lower mass distribution than metabolites characterizing milk intake (P = 4.1 × 10−4). Candidate BFIs for milk or cheese included saccharides, a hydroxy acid, amino acids, amino acid derivatives, and dipeptides. Two serum oligosaccharides, blood group H disaccharide (BGH) and Lewis A trisaccharide (LeA), specifically reflected milk intake but with high interindividual variability. The 2 oligosaccharides showed related but opposing trends: subjects showing an increase in either oligosaccharide did not show any increase in the other oligosaccharide. This result was confirmed in urine. Conclusions New candidate BFIs for milk or cheese could be identified in healthy adults, most of which were related to protein metabolism. The increase in serum of LeA and BGH after cow-milk intake in adults calls for further investigations considering the beneficial health effects on newborns of such oligosaccharides in maternal milk. The trial is registered at clinicaltrials.gov as NCT02705560.