AimThermal sensitivity of cellular metabolism is crucial for animal physiology and survival under climate change. Despite recent efforts, effects of multigenerational exposure to temperature on the metabolic functioning remain poorly understood. We aimed at determining whether multigenerational exposure to temperature modulate the mitochondrial respiratory response of Medaka fish. MethodsWe conducted a multigenerational exposure with Medaka fish reared multiple generations at 20 and 30°C (COLD and WARM fish, respectively). We then measured the oxygen consumption of tail muscle at two assay temperatures (20 and 30°C). Mitochondrial function was determined as the respiration supporting ATP synthesis (OXPHOS) and the respiration required to offset proton leak (LEAK(Omy)) in a full factorial design (COLD‐20°C; COLD‐30°C; WARM‐20°C; WARM‐30°C). ResultsWe found that higher OXPHOS and LEAK fluxes at 30°C compared to 20°C assay temperature. At each assay temperature, WARM fish had lower tissue oxygen fluxes than COLD fish. Interestingly, we did not find significant differences in respiratory flux when mitochondria were assessed at the rearing temperature of the fish (i.e., COLD‐20°C vs. WARM −30°C). ConclusionThe lower OXPHOS and LEAK capacities in warm fish are likely the result of the multigenerational exposure to warm temperature. This is consistent with a modulatory response of mitochondrial capacity to compensate for potential detrimental effects of warming on metabolism. Finally, the absence of significant differences in respiratory fluxes between COLD‐20°C and WARM‐30°C fish likely reflects an optimal respiration flux when organisms adapt to their thermal conditions.