Morning endurance training induces superior performance adaptations compared to afternoon training 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 animal's 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.