Abstract In leaves of C3 and C4 plants, stomata open during the day to favor CO2 entry for photosynthesis and close at night to prevent inefficient transpiration of water vapor. The circadian clock paces rhythmic stomatal movements throughout the diel (24–h) cycle. Leaf transitory starch is also thought to regulate the diel stomatal movements, yet the underlying mechanisms across time (key moments) and space (relevant leaf tissues) remain elusive. Here, we developed PhenoLeaks, a pipeline to analyze the diel dynamics of transpiration, and used it to screen a series of Arabidopsis (Arabidopsis thaliana) mutants impaired in starch metabolism. We detected a sinusoidal, endogenous rhythm of transpiration that overarches days and nights. We determined that a number of severe mutations in starch metabolism affect the endogenous rhythm through a phase shift, resulting in delayed stomatal movements throughout the daytime and diminished stomatal preopening during the night. Nevertheless, analysis of tissue-specific mutations revealed that neither guard-cell nor mesophyll-cell starch metabolisms are strictly required for normal diel patterns of transpiration. We propose that leaf starch influences the timing of transpiration rhythm through an interplay between the circadian clock and sugars across tissues, while the energetic effect of starch-derived sugars is usually nonlimiting for endogenous stomatal movements.