Global population growth has emphasised the need to have sustainable and alternative sources of nutrients. In this context, microalgae have emerged as a potential solution due to their rich biochemical composition, including high-quality proteins, carbohydrates, lipids, and pigments. This study investigates the variation in microalgal growth and biochemical composition over a light:dark cycle, with a focus on the night period. Batch experiments were performed with eight Chlorella vulgaris cultures over a 7-day period. On the seventh day, biomass samples were collected at four time points in four-hour intervals and stored for subsequent biochemical analyses. During the eight-hour dark period, biomass, carbohydrate, and total chlorophyll concentrations decreased by 9%, 12.5%, and 14.4%, respectively. After four hours of light exposure, these parameters increased significantly by 6%, 15.4%, and 12.7%, respectively. Total protein, carotenoids, and fatty acid contents remained relatively stable throughout the evaluated cycle, although variations were observed in the carotenoid profile. During the dark phase, zeaxanthin decreased by 38.0%, whereas violaxanthin increased by 27.2%, suggesting complementary pigment interconversion consistent with xanthophyll cycle activity. Overall, these results highlight the importance of optimising harvesting time to enhance the production of target compounds in a sustainable production of microalgal biomass.