Phytoplankton growth depends not only on mean intensity but also on the dynamics of the light supply. In surface mixed layers, phytoplankton may rapidly move between strong light and almost darkness. The nonlinear light-dependency of growth may differ between constant and fluctuating light because of the different frequency distribution of light and/or acclimation processes. The present study compares for the first time light-dependency of photosynthesis and growth of phytoplankton communities in situ under defined mixing conditions and at fixed depths. Maximum growth rates per day were not significantly different, but the growth efficiency was much higher under constant light than under fluctuating light of sub-saturating daily irradiance. Phytoplankton incubated under fluctuating light needed about three times higher mean daily irradiances to balance photosynthesis and losses than under constant light. The difference in growth efficiency was mostly caused by the different frequency distribution of underwater light, as was estimated by a photosynthesis model of sufficient temporal resolution. The present study indicates a considerable overestimation of phytoplankton growth at sub-saturating light in well-mixed water layers by the common growth measurements under constant light. This implies an underestimation of the compensation light intensities and respective overestimations of the critical mixing depths.