Intertidal microphytobenthos (MPB) are important primary producers and provide food for herbivores in soft sediments and on rocky shores. Methods of measuring MPB biomass that do not depend on the time of collection relative to the time of day or tidal conditions are important in any studies that need to compare temporal or spatial variation, effects of abiotic factors or activity of grazers. Pulse amplitude modulated (PAM) fluorometry is often used to estimate biomass of MPB because it is a rapid, non-destructive method, but it is not known how measures of fluorescence are altered by changing conditions during a period of low tide. We investigated this experimentally using in situ changes in minimal fluorescence (F) on a rocky shore and on an estuarine mudflat around Sydney (Australia), during low tides. On rocky shores, the time when samples are taken during low tide had little direct influence on measures of fluorescence as long as the substratum is dry. Wetness from wave-splash, seepage from rock pools, run-off, rainfall, etc., had large consequences for any comparisons. On soft sediments, fluorescence was decreased if the sediment dried out, as happens during low-spring tides on particularly hot and dry days. Surface water affected the response of PAM and therefore measurements used to estimate MPB, emphasising the need for care to ensure that representative sampling is done during low tide.