Early morning run-training results in enhanced endurance performance adaptations in mice.

Endurance performance exhibits time-of-day variation in both humans and rodents, peaking in the late active-phase. However, whether the timing of endurance training influences performance adaptations remains unclear. To investigate, female mice were trained 5-d/week for 6-weeks at either ZT13 or ZT22, using treadmill running at 70% of each animals maximal capacity. Endurance performance was assessed at baseline, week-3, and week-6. Secondary outcomes included blood glucose and lactate, cage activity, body composition, liver and skeletal muscle glycogen content, mitochondrial and contractile protein expression. At baseline, late-active phase (ZT22)-tested mice exhibited significantly higher endurance capacity than early-active phase (ZT13)-tested mice (P<0.05). Following 6 weeks of training, ZT13-trained mice demonstrated a greater rate of improvement, with endurance increasing by 132% (P<0.05), compared to 45% in afternoon ZT22-trained mice. By week 6, performance improved but was similar between groups (P>0.05), despite lower absolute training volumes in the ZT13 group. Both training groups reduced fat-mass (ZT13: -31%,ZT22: -32%; P<0.05 vs. control), with no differences in lean mass, food intake or muscle and liver glycogen content (P>0.05). In skeletal muscle, ZT13-trained mice were associated with increased (P<0.05) COXIV protein expression, citrate synthase activity, and shifts in MyHC isoform expression, without changes (P>0.05) in mitochondrial content. ZT13-training elicited superior performance adaptations despite lower absolute workloads, indicating enhanced training efficiency. These findings identify exercise timing as a biologically relevant factor influencing endurance adaptation and variability in exercise responses. NEW & NOTEWORTHYThis study demonstrates that endurance training in the early active phase induces greater performance adaptations than late active phase training in mice, resulting in overcoming diurnal differences in exercise performance, despite lower absolute training volumes. These findings reveal exercise timing influences training efficiency, likely via circadian regulation of skeletal muscle metabolism. This work identifies time-of-day as a biologically relevant and underappreciated variable contributing to the heterogeneity of exercise responses, even in tightly controlled preclinical models.