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Aim Recent papers have used large palaeolimnological datasets to reveal the biodiversity patterns of aquatic microorganisms. However, scant attention has been paid to the influence of time on these patterns. Where lake surficial sediment samples are used as integrals of diversity, the time interval of each sample varies according to differences in sediment accumulation rates. This paper aims to test the reliability of using lake surface sediments to measure and to compare microbial diversity when the potential influences of the species-time relationships are taken into account. Location Alpine lakes in Europe. Methods We analysed microorganism (siliceous microalgae) assemblages in three European Alpine lakes using short sediment cores (210Pb-dated) and annual sediment trap samples from 12 UK lakes. The same number of individuals was pooled for each sample 500 times to avoid sampling effort effects and to standardize species diversity estimation. The influence of time on the diversity score was assessed by simulating an increase of time span for surface sediment samples by cumulatively adding in successive sediment core samples (from the most recent to the oldest). We used species richness ( S) and the exponential of the bias-corrected Shannon entropy index (exp( Hb-c)) to estimate diversity. Results Increasing the time interval represented by a surficial sediment sample did not affect the diversity results. The estimation of diversity was similar for cumulative and non-cumulative samples. Diversity estimation was only altered in lakes experiencing high community turnover due to strong environmental forcing during the time period spanned by the cumulative sample. Main conclusions The use of surface lake sediments is suitable for estimating the average site diversity of free-living microorganisms. Diversity is integrated in a single sample and species assemblage composition is derived from microbial communities living in distinct lake microhabitats. Species remains, accumulated in a single sample over several years of environmental variability, represent a diversity integral that captures a spatio-temporal component equivalent to the γ-diversity measure. [ABSTRACT FROM AUTHOR]