<p>The gut microbiota (GM) affects host development, behavior and disease susceptibility. Biomedical research investigating GM-mediated influences on host phenotypes often involves collecting fecal samples from laboratory mice. Many environmental factors can affect the composition of the GM in mice. While efforts are made to minimize this variation, biological and technical variability exists and may influence outcomes. Here we employed a hierarchical fecal sampling strategy (that is, sequenced multiple libraries generated from multiple pellets collected from multiple mice) to quantify the effect size of biological and technical variation and to provide practical guidance for the development of microbiome studies involving laboratory mice. We found that while biological and technical sources of variation contribute significant variability to alpha- and beta-diversity outcomes, their effect size is 3–30-times lower than that of the experimental variable in the context of an experimental group with high intergroup variability. After quantifying the variability of alpha-diversity metrics at the technical and biological levels, we simulated whether sequencing multiple fecal samples from mice improves effect size in a two-group experimental design. Our simulation determined that collecting five fecal samples per mouse increased effect size, reducing the minimum number of animals per group required by 5% while dramatically increasing sequencing costs. Our data suggest that the effect size of biological and technical factors may contribute appreciable variability to an experimental paradigm with relatively low mean differences. In addition, repeated sampling improves statistical power; however, its application is probably impractical given the increased sequencing costs.</p>