**Background:** Circadian rhythms regulate cardiovascular function and metabolism, and the heart shows time-of-day dependent sensitivity to pathological hypertrophic stimuli. However, whether physiological hypertrophy induced by exercise is also temporally gated is unknown. This study aimed to determine the impact of 6 weeks of voluntary wheel running restricted to the early (EAP) or late (LAP) active period on exercise-induced cardiac adaptations in mice.
**Methods:** Male C57Bl/6J mice (n=45, 12 weeks old) were housed on a 12:12 light-dark cycle (lights off at ZT12). Mice were allocated to three groups: sedentary (SED, locked wheel), early active phase exercise (EAP, wheel access ZT12-ZT18), or late active phase exercise (LAP, wheel access ZT18-ZT24), 5 days/week for 6 weeks. Wheel activity was recorded wirelessly. After training, mice had 24-h ad libitum wheel access to assess activity rhythms. Hearts were excised at ~ZT13.5 for analysis. Outcomes included: heart weight (raw and normalized to body weight and tibia length), cardiomyocyte cross-sectional area (CSA) via histology, mRNA expression of circadian clock genes (Bmal1, Clock, Per1, Per2, Rev-erbα, Cry1, Cry2) and IGF1 (hypertrophy marker) by RT-PCR, and mitochondrial oxidative phosphorylation (OXPHOS) complex protein expression by western blot. Statistical analyses used two-way repeated-measures ANOVA, unpaired t-tests, and one-way ANOVA with Tukey's post-hoc tests.
**Key Results:**
- **Training volume:** EAP mice ran significantly greater distances than LAP mice in weeks 2-4 (e.g., week 2: EAP 8.03±0.47 km vs LAP 4.68±0.70 km, p<0.01; week 3: 8.88±0.30 vs 5.98±0.76 km, p=0.01; week 4: 9.21±0.62 vs 6.40±0.69 km, p=0.04). By weeks 5-6, distances were not significantly different.
- **Activity rhythms:** During 24-h free access, acrophase was significantly later in LAP vs EAP mice (EAP: 15.22±0.25 vs LAP: 16.74±0.19, p<0.001). Total distance was similar (EAP: 12.46±1.63 km vs LAP: 12.72±1.98 km, p=0.92). EAP mice performed 78.35±2.96% of activity in the first 6 h of the active phase vs 56.10±2.16% for LAP (p<0.01).
- **Cardiac hypertrophy:** Absolute heart weight was significantly higher in EAP vs SED (EAP: 130.90±3.2 mg vs SED: 121.4±3.4 mg, p<0.05), but not in LAP. Heart weight/body weight was higher in both EAP (4.8±0.1) and LAP (4.7±0.1) vs SED (4.5±0.1, both p<0.05). Cardiomyocyte CSA was increased in both exercise groups vs SED (SED: 158.9±6.6, EAP: 194.6±5.0, LAP: 180.0±5.4; p<0.05), with a trend for higher CSA in EAP vs LAP (p=0.08). IGF1 mRNA was elevated in both EAP (1.30±0.045) and LAP (1.50±0.07) vs SED (1.00±0.07, p<0.05).
- **Circadian clock genes:** Per1 expression was significantly higher in LAP vs SED (LAP: 2.45±0.64 vs SED: 1.00±0.50, p<0.01) and vs EAP (EAP: 1.29±0.49, p=0.02). No other clock genes showed significant differences.
- **Mitochondrial OXPHOS:** Complex V (ATP synthase) was significantly higher in LAP vs EAP (LAP: 1.27±0.11 vs EAP: 0.96±0.06, p=0.03), with no differences in Complexes I-IV.
**Clinical Implications:** This study demonstrates that the timing of exercise differentially influences cardiac adaptations in mice. Early active period (morning-equivalent) exercise preferentially promotes physiological cardiac hypertrophy, while late active period (evening-equivalent) exercise has a greater impact on circadian clock gene expression and mitochondrial Complex V. These findings suggest that exercise prescription timing could be tailored to optimize specific cardiovascular outcomes, such as maximizing hypertrophy in cardiac rehabilitation or aligning circadian rhythms. However, the study is limited by the use of male mice only, a single sacrifice time point, and potential confounding by training volume differences in early weeks. Future studies should include functional assessments (e.g., echocardiography) and investigate whether these effects translate to humans.