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Objective: The influence of different faecal collection methods on metagenomic analyses remains under discussion, and there is no general agreement on which collection method is preferable for gut microbiome research. We compared faecal samples collected in tubes without preservatives with those containing 10 mL of 96% ethanol for gut microbiome research when the timeframe from defecation to freezing at - 80 °C was up to 24 h. We aimed to compare the collection methods on faeces from participants with inflammatory and non-inflammatory gastrointestinal disorders and healthy controls to investigate the most suitable method when considering data yield, human fraction of sequencing reads, and ease of use. We also examined the faecal sample homogeneity., Results: Faeces collected in tubes without preservatives resulted in more sequencing reads compared to faeces collected in tubes with 96% ethanol and were also easier to handle. The human fraction of total reads in faeces collected in ethanol from participants with inflammatory bowel disease was higher than all other samples. DNA extraction and sequencing from two different locations in the same faecal sample gave similar results and showed sample homogeneity., Competing Interests: Declarations Ethics approval and consent to participate The study was performed in accordance with the Revised Declaration of Helsinki. The study was registered at www.clinicaltrials.gov as NCT04808271 and NCT04842149. All participants provided written informed consent to participate after verbal and written information was given. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)