Nutritional challenges have distinct impact on human muscle metabolome between morning and evening. (A) Histograms exhibit the number of significantly increased or decreased muscle metabolites by HFD (pink bar) and HCD (green bar) in the morning (top) and evening (bottom). (B–E) Venn diagrams show the overlap of the number of increased muscle metabolites by HFD (B), decreased muscle metabolites by HFD (C), increased muscle metabolites by HCD (D), and decreased muscle metabolites by HCD (E) between morning and evening. Pie charts exhibit the biological classifications of muscle metabolites exclusively altered in the morning (left), evening (right) and common to morning and evening (middle). Numbers within Venn diagrams and pie charts indicate number of changed metabolites. (F) Volcano plots exhibit the significance of altered muscle metabolite by HFD (pink) and HCD (green) in the morning. The x-axis and y-axis represent the fold change (post/pre) and significance of changed metabolites, respectively. (G) Volcano plots exhibit the significance of altered muscle metabolite by HFD (pink) and HCD (green) in the evening. (H) Top 5 muscle metabolites exclusively changed by HFD (top) and HCD (bottom) in the morning. (I) Top 5 muscle metabolites exclusively changed by HFD (top) and HCD (bottom) in the evening. ** and *** indicates P < 0.01 and P < 0.001 between pre and post, §, §§ and §§§ indicate P < 0.05, P < 0.01 and P < 0.001 between morning and evening before nutritional challenges, †, ††, ††† indicate P < 0.05, P < 0.01 and P < 0.001 between morning and evening after nutritional challenges. Two-way ANOVA compares the mean differences of metabolites levels between pre- and post-nutritional challenges. Adjusted p-value cutoff of 0.05 was used. N = 8 biological replicates in each time of day were subjected to the metabolomics analysis.