The heat liberated upon stress production in isolated cardiac muscle provides insights into the complex thermodynamic processes underlying mechanical contraction. To that end, we simultaneously measured the heat and stress (force per cross-sectional area) production of cardiac trabeculae from rats using a flow-through micromechanocalorimeter. In a flowing stream of O(2)-equilibrated Tyrode solution (∼22°C), the stress and heat production of actively contracting trabeculae were varied by 1) altering stimulus frequency (0.2-4 Hz) at optimal muscle length (L(o)), 2) reducing muscle length below L(o) at 0.2 and 2 Hz, and 3) changing extracellular Ca(2+) concentrations ([Ca(2+)](o); 1 and 2 mM). Linear regression lines were adequate to fit the active heat-stress data. The active heat-stress relationships were independent of stimulus frequency and muscle length but were dependent on [Ca(2+)](o), having greater intercepts at 2 mM [Ca(2+)](o) than at 1 mM [Ca(2+)](o) (3.5 and 2.0 kJ·m(-3)·twitch(-1), respectively). The slopes among the heat-stress relationships did not differ. At the highest experimental stimulus frequency, pronounced elevation of diastolic Ca(2+) resulted in incomplete twitch relaxation. The resulting increase of diastolic stress, which occurred with negligible metabolic energy expenditure, subsequently diminished due to the time-dependent loss of myofilament Ca(2+)-sensitivity.