Incompatibility of nitrogen fixation and oxygen production compels unicellular diazotrophic cyanobacteria to perform photosynthesis during daytime and restrict nitrogen fixation to nighttime. The marine diazotroph Cyanothece BG 043511 was grown in continuous culture under three light/dark regimes (16L : 8D, 12L : 12D, and 8L : 16D h); we monitored nitrogen fixation and potential photosynthetic efficiency simultaneously online to reveal how their temporal separation is affected by different LD regimes. An increase in nitrogen fixation rate at night coincided with a rise in pulse-amplitude modulated fluorescence, indicating that the enhanced respiratory electron transport to fuel diazotrophy affects the oxidation state of the plastoquinone pool. This may offer an alternative approach to assess instantaneous nitrogen fixation activity. Regardless of photoperiod, the maximum rate of nitrogen fixation was conserved at about 20 h after the onset of the light. Consequently, nitrogen fixation rates peaked at different moments in the dark: relatively early in the 16L : 8D cycle, at midnight in 12L : 12D, and relatively late in 8L : 16D. Under 16L : 8D, nitrogen fixation extended into the light, demonstrating the functional plasticity of nitrogen fixation in Cyanothece. Highest daily amounts of nitrogen fixed were obtained in 12L : 12D, which is consistent with the natural LD cycle of subtropical latitudes in which Cyanothece thrives.