Understanding how metabolic costs change in relation to increasing temperature under future climate changes is important to predict how ectotherms will be affected across the globe. In fish, whole body respiration is traditionally used to estimate aerobic performance via an organism’s minimum and maximum oxygen consumption rates. However, mitochondria play a crucial role in the aerobic cascade and may be a useful surrogate of aerobic performance. To test whether whole body oxygen consumption and mitochondrial capacity are correlated, we estimated whole body metabolic and mitochondrial respiration rates (using permeabilized red muscle fibres) in brook trout ( Salvelinus fontinalis (Mitchill, 1814)) at 10, 15, and 20 °C. Standard metabolic rate increased with acclimation temperature, while maximum rates were less sensitive. All mitochondrial respiration rates increased with acclimation temperature, suggesting that red muscle mitochondrial preparations may correlate to the minimal metabolic demands in this species. When expressed as relative rates of electron flow, the red muscle fibres showed no effect of temperature on mitochondrial coupling efficiency. However, there was a pattern of declining capacity to augment respiration via complex II with increasing temperature with a concomitant increase in the capacity of the phosphorylating system relative to maximal rates of mitochondrial electron flow.