ABSTRACT: This study investigates the ability to use in vivo chlorophyll a fluorescence to quantify the productive biomass of undisturbed microphytobenthic communities, defined as the photosynthetic biomass present in the photic zone of thesediment and actually contributing to measurable photosynthesis. The purposes of defining and quantifying productive biomass are (1) to evaluate the effect of the migratory rhythms on the variability of microphytobenthic photosynthesis and (2) tocharacterise the community photophysiological response independently of the migratory stage, through the estimation of biomass-specific, community-level photosynthetic rates. The possibility of using chl a fluorescence, as measurednon-destructively at the sediment surface, to trace variations in the productive biomass of microphytobenthos was confirmed by testing (1) the variability of the relationship between fluorescence emission and chl a concentration under varyingtemperature and irradiance levels and (2) the effects of natural variability in the vertical profile of chl a within the photic zone of the sediment on the depth-integrated fluorescence signal, F, measurable at the surface. Dark-levelfluorescence, F₀, was found to allow for tracing variations in chl a concentration under the range of temperature and irradiance variability found in situ. Depth-integration of fluorescence emission resulted in only afraction of total productive biomass being detectable at the surface. However, this fraction was found to be sufficiently constant to allow for the use of F₀ to proportionally follow variations in community productive biomass. Onaverage, measurements of productive biomass on natural samples overestimated by a factor of 1.30 during low tide and underestimated by a factor of 0.66 during high tide, mostly due to variations in the chl a profile in the photic zone associatedwith vertical migrations. The method was applied to intertidal microphytobenthic communities of the Tagus estuary, Portugal, by non-destructively measuring F₀ (using pulse amplitude modulated fluorometry) and photosynthesis (using oxygenmicroelectrodes) on the same samples under in situ conditions. The results showed that a significant proportion of the hourly and fortnightly variability in the community photosynthetic light response was explained solely by variations inF₀ associated with movements of microalgae, identifying migratory rhythms as the main cause for short-term variability in intertidal benthic primary productivity.