Cardiac remodelling in response to thermal acclimation has been observed in a number of fish species, with many presenting hypertrophy in response to chronic cold and the opposite in response to chronic warming. As cardiac remodelling events alter energetic requirements, the plasticity of metabolic processes may be important for successful thermal acclimation. Whilst essential in signalling pathways, Reactive Oxygen Species (ROS) can accumulate under stressful circumstances and cause oxidative damage, reduced respiration and programmed cell death. Despite their pivotal role as the major suppliers of cellular energy, comparatively few studies have investigated the effects of temperature acclimation on fish cardiac mitochondria and ROS production. Rainbow trout, Oncorhynchus mykiss, were exposed to cold (5°), control (10°) and warm (18°) temperatures for 8-12 weeks to induce long-term cardiac remodelling. Mitochondrial respiration was measured simultaneously with ROS production in ventricular homogenates using high resolution respirometry and fluorometry, respectively. An array of metabolic enzymes were assessed including enzymes from the electron transport chain (ETC) and anaerobic/fatty acid oxidation pathways. Long term exposure to cold and warm temperatures did not alter mitochondrial respiration or the activity of enzymes within the ETC. Interestingly there was also no enzymatic shift toward alternative pathways such as fatty acid oxidation or anaerobic glycolysis. Our results suggest that the metabolic processes studied within cardiac tissue are unaffected by long term temperature acclimation.