Abstract Context Alterations in the lipid metabolism are linked to metabolic disorders such as insulin resistance (IR), obesity and type 2 diabetes (T2D). Regular exercise, particularly combined training (CT), is a well-known nonpharmacological treatment that combines aerobic (AT) and resistance (RT) training benefits. However, it is unclear whether moderate-intensity exercise without dietary intervention induces changes in lipid metabolism to promote a “healthy lipidome.” Objective The study aimed to investigate the effect of 16 weeks of CT on plasma and white adipose tissue in both sexes, middle-aged individuals with normal weight, obesity (OB), and T2D using an ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS) untargeted lipidomics approach. Methods Body composition, maximum oxygen consumption (VO2max), strength, and biochemical markers were evaluated before and after the control/training period and correlated with lipid changes. CT consisted of 8 to 10 RT exercises, followed by 35 minutes of AT (45%-70% VO2max), 3 times a week for 16 weeks. Results The CT significantly reduced the levels of saturated and monounsaturated fatty acid side-chains (SFA/MUFA) in sphingolipids, glycerolipids (GL) and glycerophospholipids (GP) as well as reducing fat mass, circumferences and IR. Increased levels of polyunsaturated fatty acids in GPs and GLs were also observed, along with increased fat-free mass, VO2 max, and strength (all P < .05) after training. Conclusion Our study revealed that 16 weeks of moderate-intensity CT remodeled the lipid metabolism in OB, and T2D individuals, even without dietary intervention, establishing a link between exercise-modulated lipid markers and mechanisms that reduce IR and obesity-related comorbidities.