The application of metabolomics in nutritional research may be a useful tool to analyse and predict the response to a dietary intervention. The aim of this study was to examine metabolic changes in serum samples following exposure to an energy-restricted diet (-15% of daily energy requirements) over a period of 8 weeks in overweight and obese older adults (n = 22) using a gas chromatography/mass spectrometry (GC/MS) metabolomic approach. After 8 weeks, there were significant reductions in weight (7%) and metabolic improvement (glucose and lipid profiles). Metabolomic analysis found that total saturated fatty acids (SFAs), including palmitic acid (C16:0) and stearic acid (C18:0) and monounsaturated fatty acids (MUFAs), were significantly decreased after the 8-week intervention. Furthermore, palmitoleic acid (C16:1) was found to be a negative predictor of change in body fat loss. Both the total ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) significantly decreased, although the overall total amounts of PUFAs did not. The branched chain amino acid (BCAA) isoleucine significantly decreased in the serum samples after the intervention. In conclusion, this study demonstrated that the weight loss intervention based on a hypocaloric diet identified changes in the metabolic profiles of serum in overweight and obese older adults, with a reduction in anthropometric and biochemical parameters also found.