Morning endurance training induces superior performance adaptations compared with 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 days/wk for 6 wk at either ZT13 or ZT22, using treadmill running at 70% of each animal's maximal capacity. Endurance performance was assessed at baseline, <i>week 3</i>, and <i>week 6</i>. Secondary outcomes included blood glucose and lactate, cage activity, body composition, liver and skeletal muscle glycogen content, and 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 (<i>P</i> < 0.05). Following 6 wk of training, ZT13-trained mice demonstrated a greater rate of improvement, with endurance increasing by 132% (<i>P</i> < 0.05), compared with 45% in afternoon ZT22-trained mice. By <i>week 6</i>, performance improved but was similar between groups (<i>P</i> > 0.05), despite lower absolute training volumes in the ZT13 group. Both training groups reduced fat mass (ZT13: -31%, ZT22: -32%; <i>P</i> < 0.05 vs. control), with no differences in lean mass, food intake or muscle, and liver glycogen content (<i>P</i> > 0.05). In skeletal muscle, ZT13-trained mice were associated with increased (<i>P</i> < 0.05) cytochrome <i>c</i> oxidase subunit IV (COX IV) protein expression, citrate synthase activity, and shifts in myosin heavy chain (MyHC) isoform expression, without changes (<i>P</i> > 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.<b>NEW & NOTEWORTHY</b> This 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.