What kills animals as they approach thermal limits? Does oxygen (O2) availability limit aerobic scope, is it an intrinsic metabolic failure, or a combination of both? The heart is the most metabolically active organ, and the first to fail as ectotherms approach critical thermal limits (Tcrit). Just below Tcrit, we found that fish heart mitochondria (mt) increase O2 demands, yet respiration becomes less coupled, which should limit ATP synthesis. We explore interactions between mt O2 consumption efficiency, mt membrane potential (mtMP) and ATP dynamics as O2 supply declines. High-resolution-respirometers were coupled with fluorometric measures to assess O2 uptake, mtMP, ATP synthesis (supply) and ATP hydrolysis (demand) rates, both in “steady states”, and in the transition into anoxia. Mitochondria within permeabilised myocardium from New Zealand banded wrasse (Notolabrus fucicola) acclimated to 18oC, were examined across a range of temperatures (18-30oC), which span and exceed habitat temperatures. Importantly, we developed a novel approach to assess mt function through minimum and maximum O2 consumption rates, similar to the whole organism measure of “aerobic scope”. At low temperatures with excess O2, mt appear tightly coupled, with stable mtMP and ATP production exceeding metabolic demands. However, as temperatures increase above 25oC, these processes become impaired, with an uncoupling of oxidative phosphorylation, and increased mt respiration despite a loss of mtMP. This was reflected in an increase in mt “work” with temperature. In the transition into anoxia, mt scope and O2 binding affinity increased with temperature, however, resulted in higher non-phosphorylating respiratory rates, and declines in ATP synthesis efficiency. Mitochondrial function appears to be fully impaired at 26oC. Overall, we have developed a novel framework to assess the effects of thermal stress on mt function in the context of diminishing O2, ie mt scope.