Multivariate analyses are used widely for determining patterns of assemblage structure, inferring species–environment relationships and assessing human impacts on ecosystems. The estimation of ecological patterns often depends on sampling effort, so the degree to which sampling effort affects the outcome of multivariate analyses is a concern. We examined the effect of sampling effort on site and group separation, which was measured using a mean similarity method. Two similarity measures, the Jaccard Coefficient and Bray–Curtis Index were investigated with 1 benthic macroinvertebrate and 2 fish data sets. Site separation was significantly improved with increased sampling effort because the similarity between replicate samples of a site increased more rapidly than between sites. Similarly, the faster increase in similarity between sites of the same group than between sites of different groups caused clearer separation between groups. The strength of site and group separation completely stabilized only when the mean similarity between replicates reached 1. These results are applicable to commonly used multivariate techniques such as cluster analysis and ordination because these multivariate techniques start with a similarity matrix. Completely stable outcomes of multivariate analyses are not feasible. Instead, we suggest 2 criteria for estimating the stability of multivariate analyses of assemblage data: 1) mean within-site similarity across all sites compared, indicating sample representativeness, and 2) the SD of within-site similarity across sites, measuring sample comparability.