The effect of weight loss from long-term, mild-calorie diets (MCD) on plasma metabolites is unknown. This study was to examine whether MCD-induced weight reduction caused changes in the extended plasma metabolites. Overweight and obese subjects aged 40-59 years consumed a MCD (approximately 100 kcal/day deficit, n=47) or a weight-maintenance diet (control, n=47) in a randomized, controlled design with a three-year clinical intervention period and plasma samples were analyzed by using UPLC-LTQ-Orbitrap mass spectrometry. The three-year MCD intervention resulted in weight loss (-8.87%) and significant decreases in HOMA-IR and TG. The three-year follow-up of the MCD group showed reductions in the following 13 metabolites: L-leucine; L-phenylalanine; 9 lysoPCs; PC (18:0/20:4); and SM (d18:0/16:1). The three-year MCD group follow-up identified increases in palmitic amide, oleamide, and PC (18:2/18:2). Considering the age-related alterations in the identified metabolites, the MCD group showed a greater decrease in L-leucine, L-phenylalanine, and SM (d18:0/16:1) compared with those of the control group. Overall, the change (Delta) in BMI positively correlated with the Delta TG, Delta HOMA-IR, Delta L-leucine, and Delta SM (d18:0/16:1). The Delta HOMA-IR positively correlated with Delta TG, Delta L-leucine, Delta L-phenylalanine, and Delta SM (d18:0/16:1). The weight loss resulting from three-year mild-caloric restriction lessens the age-related increase in SM and reduces L-leucine and L-phenylalanine in overweight and obese subjects. These changes were coupled with improved insulin resistance (ClinicalTrials. gov:NCT02081898).