The intertidal zone is a dynamic habitat subject to constant tide fluctuations that provides a challenging and stressful environment for the organisms inhabiting it. As a microscopic primary producer living in association with benthic substrates, microphytobenthos play a crucial role and display adaptive characteristics like photoprotection, desiccation resistance, and migration behaviors. This vertical migration is an important adaptative mechanism that enables the microalgae constituting the microphytobenthos to optimize their light exposure and retreat into the sediments during low tide to avoid harsh conditions. Numerous studies have shown that these vertical migration mechanisms are governed by an internal clock, giving rise to endogenous rhythms. To better understand the microphytobenthic communities functioning, primary production is typically characterized through laboratory measurements, often performed independently of the photoperiod or/and tidal characteristics of the environment. However, the question arises: does the time of day and tidal phase have an impact on the processes measured? To investigate this, we simulated an artificial tidal cycle, creating four different scenarios to test the effects of ‘day hour’ and ‘tidal phase moment’. During these scenarios, we used two complementary measurement tools: a pulse-amplitude modulated (PAM) fluorometer to assess surface microphytobenthic biomass and oxygen microsensors to obtain vertical distribution of photosynthetic activity. Our study, while confirming that the endogenous rhythm of microphytobenthos can be maintained for up to three days in the laboratory, shows that the intensity of vertical migratory rhythm varies with the day hour. Importantly, we have shown that the day hour significantly influences primary production estimates, with maximum production values potentially being up to three times higher when estimated during afternoon emersions compared to nocturnal emersions. In addition, the timing of measurements in relation to the immersion/emersion cycle can also have an impact on primary production estimates, and must be considered if reliable and realistic estimates are to be obtained.