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Introduction Core diurnal clock genes regulate inflammation and tissue regeneration, yet their role in pulpal healing remains unexplored. This study examined whether expression levels of CLOCK, BMAL1, PER1 and PER2 are associated with outcomes of vital pulp therapy (VPT) performed at different times of day.Methods 60 healthy patients (18–40 years) requiring VPT were enrolled in this single-centre prospective observational study. Participants were stratified by treatment time: morning (08:00–12:00), afternoon (12:00–16:00) and evening (16:00–20:00) (n=20/group). Oral fluid and pulp tissue samples were collected for real-time quantitative PCR, with expression of CLOCK, BMAL1, PER1 and PER2 normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Delta Ct (ΔCt). Clinical and radiographic follow-up was conducted at 1, 3, 6 and 12 months.Results CLOCK expression (ΔCt) was significantly higher in the morning (2.1±0.6) compared with the afternoon (3.3±0.7) and evening (4.5±0.5) groups (p<0.001). BMAL1 showed a similar pattern (1.8±0.5 vs 2.7±0.6 vs 3.9±0.4; p<0.001). 12-month success rates were 90% (morning), 75% (afternoon) and 60% (evening) (χ² = 6.80, p=0.034). CLOCK (r = –0.72) and BMAL1 (r = –0.69) strongly correlated with healing outcomes (p<0.01). Logistic regression confirmed CLOCK (OR=2.8, 95% CI 1.5 to 5.4, p=0.002) and BMAL1 (OR=2.3, 95% CI 1.2 to 4.8, p=0.009) expression as independent predictors of VPT success (Nagelkerke R² = 0.56). PER1 showed minimal diurnal variation, and PER2 was not significantly associated with treatment success.Conclusions Morning procedures are associated with higher CLOCK and BMAL1 expression and improved VPT outcomes, supporting the potential of chronobiology-informed, time-optimised endodontic therapy.