Metabolomic analyses of fecal material are gaining increasing attention because the gut microbial ecology and activity have an impact on the human phenotype and regulate host metabolism. Sample preparation is a crucial step, and in this study we recommend a methodology for extraction and analysis of fresh feces by NMR-based metabolomics. The evaluation of extraction solvents showed that buffer extraction is a suitable approach to extract metabolic information in feces. So, the effects of weight-to-buffer (Wf:Vb) combinations and the effect of sonication and freeze-thaw cycles on the reproducibility, chemical shift variability, and signal to noise ratio (SNR) of the 1H NMR spectra were evaluated. Based on our results, we suggest that fresh fecal extraction with a Wf:Vb ratio of 1:2 may be the optimum choice to determine the overall metabolite composition of feces. In fact, more than 60 metabolites have been assigned in the NMR spectra obtained from the fresh fecal buffer extract, and assignment of the lipophilic signals is also presented. To our knowledge, some of the metabolites are reported here for the very first time employing 1H NMR spectroscopy on human fecal extracts.