Introduction <p>Faecal metabolomics investigates small molecules in stool to elucidate metabolic pathways and identify potential biomarkers. However, topographical heterogeneity within individual stool specimens poses a challenge for data consistency. Although this issue is well recognised, quantitative comparisons across defined stool regions, sampling depths, and whole-stool homogenates remain limited.</p> Objectives <p>This pilot study aimed to (i) quantify metabolite variation across five regions (from head to tail) and different depths of the faecal bulk, and (ii) determine whether spot sampling can serve as a representative alternative to whole-stool homogenisation for faecal metabolite profiling by proton nuclear magnetic resonance (¹H NMR) spectroscopy.</p> Methods <p>Seventy-six metabolites were quantified across defined faecal regions and depths. Comparative analyses determined spatial differences in metabolite abundance and assessed the similarity between spot samples and bulk homogenates.</p> Results <p>Distinct metabolic profiles were observed between the two ends of the faecal bulk. The tail region, representing more recently discharged material, exhibited higher levels of 3-(3-hydroxyphenyl)propionate (+ 57–58%) and 3-phenylpropionate (+ 50%), whereas the head region showed greater abundance of proteolysis-related metabolites such as isovalerate (+ 30%) and methionine (+ 54%). No consistent differences were detected across sampling depths. Despite these metabolite-specific differences, overall deviations from the homogenised profile were small, with mean absolute differences remaining approximately 1% or less across regions.</p> Conclusion <p>Pronounced longitudinal heterogeneity exists in faecal metabolite composition along the stool axis. In this pilot cohort, tail-region spot sampling most closely represents the overall faecal metabolome and may serve as a practical, less labour-intensive alternative to whole-stool homogenisation for metabolomic studies.</p>