 rate (FDR) are presented in [Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}. Interestingly, the metabolites were predominantly increased following freezing at −80 °C prior to preparation of fecal water ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}). The sample stored on ice for 24 h had the highest fidelity to the freshly prepared samples. Closer examination of the significantly different metabolites revealed that the BCAAs, aromatic amino acids, Krebs cycle intermediates, and monosaccharides were found at higher levels in fecal water prepared from frozen samples. However, the short chain fatty acid (SCFA), butyrate, was lower in fecal water from frozen/defrosted, and on ice samples. [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"} shows the typical spectra of fecal water analyzed from fresh, frozen, and on ice samples.

![Box plots of significant metabolites from the resulting analysis of fecal water from fresh, frozen/defrosted, and on ice samples. \* denotes significant differences between frozen and fresh samples and frozen and on ice samples (Bonferroni post hoc). \# denotes significant difference between fresh and frozen sample and fresh and on ice samples (Bonferroni post hoc). All *p*-values were FDR adjusted.](ao-2018-01761t_0002){#fig2}

![600 MHz ^1^H NMR spectra of fecal water colored by sample storage: analysis of fecal water from fresh (blue), frozen (red), and on ice (green) samples. Assignations of significant metabolites (FDR \< 0.05) are presented. BCAAs: valine, leucine, and isoleucine.](ao-2018-01761t_0003){#fig3}

###### Differential Fecal Metabolites between the Different Conditions of Sample Storage[a](#t2fn1){ref-type="table-fn"}

  metabolite      *P*-value   FDR     description
  --------------- ----------- ------- ----------------------------------------------------------
  aspartate       \<0.001     0.008   Krebs cycle: oxaloacetate transamination
  butyrate        \<0.001     0.017   short chain fatty acid
  fructose        \<0.001     0.008 