Determining the mechanisms underpinning the thermal limits of organisms is essential to anticipate the impact of climate change on their survival. As mitochondrial activity is key for cellular ATP production, we aimed at investigating the link between mitochondrial dysfunction and the upper thermal limit (CT max ) at which neuromuscular functions are lost. We reared medaka fish (Oryzias latipes) over generations at 20 • C (COLD) and 30 • C (WARM) during five years and measured their CT max and their mitochondrial oxygen consumption rates (OCRs) at 25 • C and at their CT max . We found that multigenerational exposure at elevated temperature increases the CT max by 13 %. We observed for all fish a general decrease in the OCRs and for the RCR of complex-I between 25 • C and their CT max suggesting a link between CT max and cerebral mitochondrial performance. Finally, we found an increase in the contribution of the mitochondria complex II to OCRs at elevated temperature suggesting that this complex underlie flexibility in mitochondrial functioning at high temperature. Our study highlights the link between whole organism thermal tolerance and mitochondrial dysfunction and that multigenerational exposure can modify mitochondrial and thermal performance.