Background
24h whole-body substrate metabolism and the endogenous circadian clock operative in human skeletal muscle are compromised upon metabolic diseases. The aim of this study was to compare the skeletal muscle metabolome derived from young healthy (YH) vs. older, metabolically-compromised men (OMC) by serial muscle sampling performed under 24h real-life conditions to explore differences in the temporal dynamics of muscle metabolism.

Methods
Under standardized continuous laboratory conditions, five sequential skeletal muscle biopsies were obtained from 12 YH (22±2 years, BMI: 22.4±2.2 kg/m2) and 12 OMC (65±9 years, BMI: 30.3±2.7 kg/m2) every 5 hours between 8AM and 4AM, while three meals were provided during daytime. Muscle metabolites were determined using UPLC/HRMS-based semi-targeted metabolomics. Abundance levels of metabolites were compared between groups, metabolites were analysed for 24h-rhythmicity per group and rhythmic properties were compared between groups.

Results
Metabolites associated with the initial steps of glycolysis and the end-product of hexosamine biosynthesis were higher in OMC around the clock, whereas metabolites associated with the urea cycle, glutamine-alpha-ketoglutarate, ketone and redox metabolism were lower in OMC. The largest number of differently expressed metabolites was found during the night at 4AM. Both groups demonstrated 24h-rhythmicity in ~50% of detected metabolites but with only partial overlap between groups. From the total of 115 detected metabolites, 15 metabolites were only rhythmic in YH, such as adenosine, and 17 metabolites only in OMC. Cis-aconitate, FAD, and UDP where phase shifted in OMC, hydroxybutyrate and hippuric acid displayed a reduced 24h-rhythm amplitude in OMC compared to YH.

Conclusion
Large divergence in the skeletal muscle metabolome is evident around the clock between the edges of the metabolic health spectrum with greatest differences during the night. Temporal changes within the metabolome could contribute to the compromised 24h whole-body substrate metabolism and disturbed circadian clock in skeletal muscle of metabolically-compromised humans.