Consistency in studies of feline and canine gut microbiomes relies heavily on how faecal samples are collected and preserved. This study evaluated practical strategies for sampling and storing cat and dog faeces, suitable for both laboratory and home settings. We specifically examined whether storing samples at room temperature for up to 12 hours and collecting material from different parts of the stool affected microbial diversity, taxa composition, or DNA quality. Faecal samples were collected from 10 healthy cats and 10 healthy dogs, maintained at 20 °C, and subsampled from the initial, middle, and terminal portions of each stool, at either surface or core, at multiple time points (0–12 h), before stabilization at −80 °C. DNA extraction was performed using Illumina NovaSeq sequencing. Alpha diversity was comparable between canine and feline samples, with Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria being the dominant phyla. Prevotella predominated in both species, while Fusobacterium was absent in cats. Room-temperature storage for up to 12 hours generally did not significantly affect alpha diversity, taxonomic composition, or DNA yield. Sampling location within the stool had minimal effect, although surface samples from cat faeces after 12 hours exhibited increased alpha diversity, with a similar but non-significant trend in dogs. Beta diversity analyses revealed that individual differences were the primary determinant of microbial composition (R² = 0.64 in dogs, 0.88 in cats), while sampling time and site had smaller but detectable influences. Cat and dog faeces can be stored at room temperature for up to 12 hours without major alterations in microbial composition or DNA recovery. For samples stored longer than 6 hours, core sampling is recommended over surface sampling to maintain representative microbial profiles.
