Changes in eating habits, food composition and processing are involved in the ''nutritional transition'' that accompanied the obesity pandemic and the burst of metabolic diseases. This study is one of the first to describe the metabolic trajectories that differentiate the responses of overweight (OW) from lean individuals during weight gain. Nineteen lean and 19 OW male volunteers were submitted to moderate weight gain using a lipid-enriched overfeeding protocol designed to add about 3,300 kJ per day in excess to their usual diet. Metabolic explorations in combination with plasma and urine metabolomic profiles using liquid chromatography coupled with mass spectrometry were determined along 8 weeks to compare metabolic trajectories and determine early changes in metabolic processes after identification of specific early responding markers. Urinary metabolomic profiles during overfeeding evidenced differences in metabolic trajectories between groups, characterized by an increase over time of short-, medium-chain acylcarnitines, and bile acids in overweight subjects. For most of the anthropometric, metabolic parameters and plasma metabolomics data, the time-course evolution of all subjects was similar with distinction between groups. Plasma abundances of unsat-urated lysophosphosphatidylcholine (22:6) decreased over time more importantly in normal weight subjects while most of those of the saturated species increased in both groups. These findings not evidenced with classical parameters, indicate a differential response to overfeeding in urine metabolomes of subjects, suggesting different nutrient metabolic fate with weight status. Subtle plasma and urine metabolic changes, mostly related to differences in the adaptation of b-oxidation and inflammation indicate a lower metabolic flexibility of OW subjects facing weight gain induced by overfeeding. Keywords Nutritional metabolomics Á UPLC–MS Á Urinary metabolic trajectories Á Weight gain Á Lipid-enriched overnutrition