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Residual material from fecal immunochemical test (FIT) tubes, commonly used in colorectal cancer screening programs, offers a valuable resource for large-scale gut microbiome studies. With recent advances in sequencing technologies, sequencing the full-length bacterial 16S ribosomal gene is now feasible. In this study, we evaluated the impact of pre-analytical handling conditions on microbiome profiling using FIT samples. Stool samples from eight healthy adults were subjected to various short-term (+ 20 °C) and long-term (-18 °C or -80 °C) storage conditions prior to DNA extraction. We also investigated the effects of sampling variation and the presence of buffer medium. Full-length 16S rRNA gene amplicons were generated and sequenced using Oxford Nanopore Technology to characterize the microbiome composition. Despite variations in sampling and storage conditions, microbiome richness, Shannon diversity, and individual characteristics were preserved, demonstrating the robustness of microbiomes extracted from FIT tubes. However, some variations were noted, such as increased amounts of collagenase-producing bacteria from 0.2 to 0.6% to 1.7-2.6% in samples stored at +20 °C for 4-10 days. Despite unsupervised and varying sampling and storage conditions, the fecal 16S rRNA microbiomes remained representative and robust. These findings support the usability of FIT samples for large-scale population microbiome research.