Aims/hypothesisMetabolic effects of exercise may partly depend on the time-of-day when exercise is performed. We tested the hypothesis that exercise timing affects the adaptations in multi-tissue metabolome and skeletal muscle proteome profiles in men with type 2 diabetes. MethodsMen fitting the inclusion (type 2 diabetes, age 45–68 years and body mass index 23–33 kg/m2) and exclusion criteria (insulin treatment, smoking, concurrent systemic disease, and regular exercise training) were included in a randomized crossover trial (n = 15). Participants included in this metabolomics and proteomics analysis fully completed all exercise sessions (n = 8). The trial consisted of two weeks of high-intensity interval training (HIT) (three sessions/week) either in the morning (08:00, n = 5) or afternoon (16:45, n = 3), a two-week wash-out period, and an additional two weeks of HIT at the opposing time. Participants and researchers were not blinded to group allocation. Blood, skeletal muscle and subcutaneous adipose tissue were obtained before the first, and after each training period. Broad-spectrum, untargeted proteomic analysis was performed on skeletal muscle, and metabolomic analysis was performed on all biosamples. Differential content was assessed by linear regression and pathway set enrichment analyses were performed. Coordinated metabolic changes across tissues were identified by Spearman correlation analysis. ResultsMetabolic and proteomic profiles remained stable after two weeks of HIT, and individual metabolites and proteins were not altered, irrespective of the time of day at which the training was performed. However, coordinated changes in relevant metabolic pathways and protein categories were identified. Morning and afternoon HIT similarly increased plasma diacylglycerols, skeletal muscle acyl-carnitines, and subcutaneous adipose tissue sphingomyelins and lysophospholipids. Acyl-carnitines were central to training-induced metabolic cross-talk across tissues. Plasma carbohydrates, via the penthose phosphate pathway, were increased and skeletal muscle lipids were decreased after morning compared to afternoon HIT. Skeletal muscle lipoproteins were higher, and mitochondrial complex III abundance was lower after morning compared to afternoon HIT. Conclusions/interpretationWe provide a comprehensive analysis of a multi-tissue metabolomic and skeletal muscle proteomic responses to training at different times of the day in men with type 2 diabetes. Increased circulating lipids and changes in adipose tissue lipid composition were common between morning and afternoon HIT. However, afternoon HIT increased skeletal muscle lipids and mitochondrial content to a greater degree than morning training. Thus, there is a diurnal component in the metabolomic and proteomic response to exercise in men with type 2 diabetes. The clinical relevance of this response warrants further investigation.