The circadian clock is a molecular timer of metabolism that affects the diurnal pattern of stomatal conductance (gs), amongst other processes, in a broad array of plant species. The function of circadian gs regulation remains unknown and here, we test whether circadian regulation helps to optimize diurnal variations in stomatal conductance. We subjected bean (Phaseolus vulgaris) and cotton (Gossypium hirsutum) canopies to fixed, continuous environmental conditions of photosynthetically active radiation, temperature, and vapour pressure deficit (free-running conditions) over 48 h. We modelled gs variations in free-running conditions to test for two possible optimizations of stomatal behaviour under circadian regulation: (i) that stomata operate to maintain constant marginal water use efficiency; or (ii) that stomata maximize C net gain minus the costs or risks of hydraulic damage. We observed that both optimization models predicted gs poorly under free-running conditions, indicating that circadian regulation does not directly lead to stomatal optimization. We also demonstrate that failure to account for circadian variation in gs could potentially lead to biased parameter estimates during calibrations of stomatal models. More broadly, our results add to the emerging field of plant circadian ecology, where circadian controls may partially explain leaf-level patterns observed in the field.