Changes in eating habits, food composition andprocessing are involved in the ‘‘nutritional transition’’ thataccompanied the obesity pandemic and the burst of metabolicdiseases. This study is one of the first to describe themetabolic trajectories that differentiate the responses ofoverweight (OW) from lean individuals during weightgain. Nineteen lean and 19 OW male volunteers weresubmitted to moderate weight gain using a lipid-enrichedoverfeeding protocol designed to add about 3,300 kJ perday in excess to their usual diet. Metabolic explorations incombination with plasma and urine metabolomic profilesusing liquid chromatography coupled with mass spectrometry were determined along 8 weeks to comparemetabolic trajectories and determine early changes inmetabolic processes after identification of specific earlyresponding markers. Urinary metabolomic profiles duringoverfeeding evidenced differences in metabolic trajectoriesbetween groups, characterized by an increase over time ofshort-, medium-chain acylcarnitines, and bile acids inoverweight subjects. For most of the anthropometric,metabolic parameters and plasma metabolomics data, thetime-course evolution of all subjects was similar withdistinction between groups. Plasma abundances of unsaturatedlysophosphosphatidylcholine (22:6) decreased overtime more importantly in normal weight subjects whilemost of those of the saturated species increased in bothgroups. These findings not evidenced with classicalparameters, indicate a differential response to overfeedingin urine metabolomes of subjects, suggesting differentnutrient metabolic fate with weight status. Subtle plasmaand urine metabolic changes, mostly related to differences in the adaptation of b-oxidation and inflammation indicatea lower metabolic flexibility of OW subjects facing weightgain induced by overfeeding.