Photosynthetic parameters of a microphytobenthic (MPB) biofilm grown in a tidal mesocosm were measured on undisturbed sediment using variable fluorescence-based measurements of electron transport rate (ETR), as well as by ETR and 14C assimilation measurements in optically thin suspensions of algal cells. Absorption cross-sections of the MPB suspensions were quantified using the quantitative filter technique and by reconstruction using HPLC-derived pigment concentrations. Photosynthetic parameters derived by the 3 methods were compared on 3 days, representing different biofilm growth/[chl a] conditions, at the start, middle and end of the daytime tidal emersion. Comparisons of ETR and radioisotope-derived photosynthetic parameters measured on optically thin suspensions were not significantly different, confirming that with an appropriate estimation of the irradiance absorbed by Photosystem II (PSII), under optically well-defined conditions, variable fluorescence is a reliable measure of MPB photosynthetic rates. In contrast, significant differences of up to 60% were observed between the maximum photosynthetic capacity (PBmax) measured on undisturbed sediment and in suspensions. These differences were observed at high [chl a] (coinciding with low growth rates) towards the end of emersion periods. Comparison of the effective quantum efficiency (ΔF/Fm’) at the highest light steps of photosynthetic-irradiance (P-E) curves suggested that the overestimation was due to the poor definition of the complex sediment optics, which interacted presumably with photo-taxis and/or single species migrations. Definition of the optics within undisturbed sediments, particularly considering the complex effects of migration, is a serious challenge, limiting the application of variable fluorescence techniques in situ on undisturbed sediments.