Using a high sensitivity differential scanning calorimeter in isothermal mode, we directly measured heat production in eukaryotic protists from 5 phyla spanning over 5 orders of magnitude in carbon biomass and 8 orders of magnitude in cell volume. Our results reveal that metabolic heat production normalized to cell mass is virtually constant in these organisms, with a median of 0.037 pW pg C(-1) (95% confidence interval = 0.022-0.061 pW pg C(-1)) at 5 degrees C. Contrary to allometric models, the relationship between heat production and cell carbon content or surface area is isometric (scaling exponents, 1.056 and 1.057, respectively). That heat production per unit cell surface area is constant suggests that heat flux through the cell surface is effectively instantaneous, and hence that cells are isothermal with their environment. The results further suggest that allometric models of metabolism based on metazoans are not applicable to protists, and that the underlying metabolic processes in the latter polyphyletic group are highly constrained by evolutionary selection. We propose that the evolutionary constraint leading to a universally constant heat production in single-celled eukaryotes is related to cytoplasmic packaging of organelles and surface area to volume relationships controlling diffusion of resources to these organelles.