The study of the fecal metabolome is an important area of research to better understand the human gut microbiome and its impact on human health and diseases. However, there is a lack of work in examining the impact of storage and processing conditions on the metabolite levels of fecal water. Furthermore, there is no universal protocol used for the storage of fecal samples and preparation of fecal water. The objective of the current study was to examine the impact of different storage conditions on fecal samples prior to metabolite extraction. Fecal samples obtained from nine healthy individuals were processed under different conditions: (1) fresh samples prepared immediately after collection, (2) fecal samples stored at 4 °C for 24 h prior to processing, and (3) fecal samples stored at −80 °C for 24 h prior to processing. All samples were analyzed using NMR spectroscopy, multivariate statistical analysis, and repeated measures ANOVA. Samples which were frozen at −80 °C prior to extraction of the metabolites exhibited an increase in the number of metabolites including branched-chain amino acids, aromatic amino acids, and tricarboxylic acid cycle intermediates. Storage of fecal samples at 4 °C ensured higher fidelity to freshly processed samples leading to the recommendation that fecal samples should not be frozen prior to extraction of fecal water. Furthermore, the work highlights the need to standardize sample storage of fecal samples to allow for the accurate study of the fecal metabolome. European Commission Horizon 2020 European Research Council