While the timing of food intake is important, it is unclear whether the effects of exercise on energy metabolism are restricted to unique time windows. As circadian regulation is key to controlling metabolism, understanding the impact of exercise performed at different times of the day is relevant for physiology and homeostasis. Using high-throughput transcriptomic and metabolomic approaches, we identify distinct responses of metabolic oscillations that characterize exercise in either the early rest phase or the early active phase in mice. Notably, glycolytic activation is specific to exercise at the active phase. At the molecular level, HIF1a, a central regulator of glycolysis during hypoxia, is selectively activated in a time-dependent manner upon exercise, resulting in carbohydrate exhaustion, usage of alternative energy sources, and adaptation of systemic energy expenditure. Our findings demonstrate that the time of day is a critical factor to amplify the beneficial impact of exercise on both metabolic pathways within skeletal muscle and systemic energy homeostasis.

Key points This is the first long‐term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. Abstract Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle‐aged, obese, insulin‐resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo‐controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high‐resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady‐state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin‐resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people.

Time-restricted feeding (TRF) improves metabolism independent of dietary macronutrient composition or energy restriction. To elucidate mechanisms underpinning the effects of short-term TRF, we investigated skeletal muscle and serum metabolic and transcriptomic profiles from 11 men with overweight/obesity after TRF (8 h day−1) and extended feeding (EXF, 15 h day−1) in a randomised cross-over design (trial registration: ACTRN12617000165381). Here we show that muscle core clock gene expression was similar after both interventions. TRF increases the amplitude of oscillating muscle transcripts, but not muscle or serum metabolites. In muscle, TRF induces rhythmicity of several amino acid transporter genes and metabolites. In serum, lipids are the largest class of periodic metabolites, while the majority of phase-shifted metabolites are amino acid related. In conclusion, short-term TRF in overweight men affects the rhythmicity of serum and muscle metabolites and regulates the rhythmicity of genes controlling amino acid transport, without perturbing core clock gene expression.