The biomass composition, namely the fatty composition, of microalgae used in aqua feed is of great importance for the nutritional value of the biomass. Day:night cycles of light and temperature could influence the growth and biomass composition of microalgae. To study the effect of these cycles on Rhodomonas sp. the algae were grown under 16:8 day:night cycles in lab-scale photobioreactors running as turbidostat. Different temperature and light intensities were applied during the light phase. Synchronized cell cycles were observed for Rhodomonas sp. under day:night conditions with oscillating cell size, cell number, biomass concentration and fatty acid content and composition. Cells increased in size during the light phase, storing energy, with cell division scheduled in the dark phase.The introduction of a 16:8 day:night cycle did not affect the biomass yield on light, when operating at optimal conditions of light (150 μmol m−2 s−1) and temperature (21 °C). However, under high light (600 μmol m−2 s−1) or temperature (25 °C), an increased biomass yield on light of up to 22% was found under day:night cycles in comparison to continuous conditions under equal light and temperature levels. Implementation of a day:night cycle increased the maximum daytime temperature for Rhodomonas sp. from 25 °C to 30 °C. The fatty acid content and composition was influence by the implementation of day:night cycles. Daily fluctuations in total fatty acid content from 76 ± 2 mg gDW−1 at the end of the light phase to 94 ± 2 mg gDW−1 in the first hours of the light phase are found. The eicosapentaenoic acid and docosahexaenoic acid content fluctuated by 30% (12.1–16.1 mgEPA+DHA gDW−1) on a daily basis. These daily fluctuations can be exploited in aqua feed applications by selecting a specific time of the day to harvest algae.