Reprogramming of the daily variation of serum metabolome in human participants by nutritional challenges. (A) Number of serum metabolites affected by diet or time. (B) Separation of samples based on the timing of the collection of serum by PCA. (C) Heatmaps display significantly gradient serum metabolites exclusively in pre- (left panel) and post-HFD (middle panel), and in both pre- and post-HFD (right panel). (D) Pie chart shows the number and ratio of significantly gradient metabolites in the serum under HFD condition. Numbers within the pie chart indicate number of gradient metabolites. Gradient, metabolites significantly different between morning and evening, Morning, metabolites being significantly higher in the morning, and Evening, metabolites being significantly higher in the evening. (E) Heatmaps display gradient serum metabolites exclusively in pre- (left panel) and post-HCD (middle panel), and in both pre- and post-HCD (right panel). (F) Pie chart exhibits the number of gradient metabolites in the serum under HCD treatment. (G) Volcano plot exhibits the significance of gradient serum metabolites under pre- (open circle) and post- (closed circle) HFD condition. The x-axis and y-axis represent the fold change (evening/morning) and significance of gradient serum metabolites, respectively. (H) Top 3 gradient serum metabolites unique to pre-HFD (top) and post-HFD (bottom). *** indicates P < 0.001 between morning and evening, § and §§ indicate P < 0.05 and P < 0.01 between pre and post in the morning, †† and ††† indicate P < 0.01 and P < 0.001 between pre and post in the evening. (I) Volcano plot exhibits the significance of gradient serum metabolites under pre- (open circle) and post- (closed circle) HCD condition. (J) Top 3 gradient serum metabolites unique to pre-HCD (top) and post-HCD (bottom). *** indicates P < 0.001 between morning and evening, § and §§§ indicate P < 0.05 and P < 0.001 between pre and post in the morning, †, ††, ††† indicate P < 0.05, P < 0.01 and P < 0.001 between pre and post in the evening. (K) Histogram shows the biological classification of gradient metabolites in the serum under HFD (left) and HCD (right) conditions. Orange and blue bars indicate the number of metabolites being higher in the morning and evening, respectively, within each classification. Two-way ANOVA compares the mean differences of metabolites levels between morning and evening. Adjusted p-value cutoff of 0.05 was used. N = 8 biological replicates in each time of day were subjected to the metabolomics analysis.