Background:Inferior quality of biological material compromises data, slows discovery, and wastes research funds. The gut microbiome plays a critical role in human health and disease, yet little attention has been given to optimizing collection and processing methods of human stool.Methods:We collected the entire bowel movement from 2 healthy volunteers: one to examine stool sample heterogeneity and one to test stool sample handling parameters. Sequencing and bioinformatic analyses were used to examine the microbiome composition.Results:The microbiome profile varied depending on where the subsample was obtained from the stool. The exterior cortex of the stool was rich in specific phyla and deficient in others while the interior core of the stool revealed opposite microbiome profiles. Sample processing also resulted in varying microbiome profiles. Homogenization and stabilization at 4°C gave superior microbial diversity profiles compared to the fresh or frozen subsamples of the same stool sample. Bacterial proliferation continued in the fresh subsample when processed at ambient temperature.Bacteroidetesproliferated andFirmicutesdiminished during the 30-minute processing of fresh sample. The frozen sample had good overall diversity but Proteobacteria diminished likely because of the freeze/thaw.Conclusion:The microbiome profile is specific to the section of the stool being sampled. Stool sample collection, homogenization and stabilization at 4°C for 24 hours provides a neat, high-quality sample of sufficient quantity that can be banked into aliquots with nearly identical microbial diversity profiles. This collection pipeline is essential to accelerate our understanding of the gut microbiome in health and disease.