The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wildtype mice on either a regular chow (RCD) or high fat diet (HFD) across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ob mice on RCD at 14-months, but was detectable at 3-months on the HFD. Liquid chromatography mass spectrometry (LC-MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither lipid classes were associated with mitochondrial dysfunction. However, a targeted LC-MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ob mice on RCD maintained similar respiratory capacity to wildtype until a late stage in ageing. However, on a HFD, unlike wildtype mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage.