AbstractDue to ease of acquisition, fecal samples are often used in studies investigating gut microbiota. Improper handling of these samples can lead to bacterial growth and alter bacterial composition. While freezing samples at −80 °C is considered gold standard, this is not suitable for studies utilizing self-sampling by lay participants or field studies. Thus to effectively prevent bacterial growth, techniques that allow efficient fecal storage outside laboratory facilities are needed. Fecal samples were collected from three donors. From each donor feces, 45 samples were collected and stored either freshly frozen at −80 or −20 °C, or in three separate storage buffers at room temperature or 4 °C for 24 or 72 hours. Bacterial composition was analyzed using Illumina amplicon sequencing of the V4 region of the 16 S rRNA gene. While storage conditions did affect bacterial composition and diversity compared to storage at −80 °C, the variation between donors superseded the variations introduced by storage. Samples stored at −20 °C most closely resembled those stored at −80 °C. When investigating variations in bacterial composition between separate study populations, fecal samples can efficiently be stored in −20 °C freezers or in one of the presented storage buffers, without severe alterations in bacterial composition.