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Leaf respiration in the dark (Rdark ) is often measured at a single time during the day, with hot-acclimation lowering Rdark at a common measuring temperature. However, it is unclear whether the diel cycle influences the extent of thermal acclimation of Rdark , or how temperature and time of day interact to influence respiratory metabolites. To examine these issues, we grew rice at 25/20°C (day/night), 30/25°C and 40/35°C, measuring Rdark and changes in metabolites at five time-points spanning a single 24-hour period. Rdark differed among the treatments and with time of day. However, there was no significant interaction between time and growth temperature, indicating that the diel cycle does not alter thermal acclimation of Rdark . Amino acids were highly responsive to the diel cycle and growth temperature, and many were negatively correlated with carbohydrates and with organic acids of the tricarboxylic acid (TCA) cycle. Organic TCA intermediates were significantly altered by the diel cycle irrespective of growth temperature, which we attribute to light-dependent regulatory control of TCA enzyme activities. Collectively, our study shows that environmental disruption of the balance between respiratory substrate supply and demand is corrected for by shifts in TCA-dependent metabolites.