Epidemiological and experimental evidence suggests that a sub-optimal environment during perinatal life programs offspring susceptibility to the development of metabolic syndrome and Type 2 diabetes. We hypothesized that the lasting impact of perinatal protein deprivation on mitochondrial fuel oxidation and insulin sensitivity would depend on the time-window of exposure. To improve our understanding of underlying mechanisms, an integrative approach was used, combining the assessment of insulin sensitivity and untargeted mass spectrometric-based metabolomics in the offspring. A hyperinsulinemic-euglycemic clamp was performed in adult male rats born from dams fed a low protein diet during gestation and/or lactation, and subsequently exposed to a Western diet (WD) for 10 weeks. Metabolomics was combined with targeted acylcarnitine profiling and analysis of liver gene expression to identify markers of adaptation to WD that influence the phenotype outcome evaluated by body composition analysis. At adulthood offspring of protein-restricted dams had impaired insulin secretion when kept under standard diet. Moreover, rats who demonstrated catch-up growth at weaning displayed higher gluconeogenesis and branched-chain amino acid catabolism, and lower fatty acid β-oxidation compared to control rats. Post-weaning exposure of IUGR-born rats to a WD exacerbated incomplete fatty acid β-oxidation and excess fat deposition. Control offspring nursed by protein-restricted mothers showed peculiar low fat accretion through adulthood and preserved insulin sensitivity even after WD-exposure. Altogether our findings suggest testable hypothesis about how maternal diet might influence metabolic outcomes (insulin sensitivity) in the next generation such as mitochondrial overload and/or substrate oxidation inflexibility dependent on the time-window of perinatal dietary manipulation.