Abstract
<title>Abstract</title> <p> Background Disrupted behavioral rhythms have been linked to adverse health outcomes, but their association with biological age acceleration remains unclear. We examined the association between a multidimensional behavioral rhythm score and biological age acceleration and explored the mediating roles of serum 25-hydroxyvitamin D3 [25(OH)D3] and insulin. Methods This cross-sectional study included 4,702 adults aged ≥ 20 years from the 2011–2014 National Health and Nutrition Examination Survey (NHANES). The behavioral rhythm score integrated sleep rhythm, dietary rhythm, and physical activity regularity. Biological age was estimated using Phenotypic Age (PhenoAge), and Phenotypic age acceleration (PhenoAgeAccel) was calculated as the residual from regressing PhenoAge on chronological age. Survey-weighted multivariable linear regression, sensitivity, subgroup, interaction, and exploratory mediation analyses were performed. Results Higher behavioral rhythm scores were associated with lower PhenoAgeAccel after multivariable adjustment. Compared with participants scoring 0–1, those scoring 2 and 3 had lower PhenoAgeAccel (score 2: β = −0.68, 95% CI, − 1.04 to − 0.31, <italic>P</italic> < 0.01; score 3: β = −0.58, 95% CI: −1.13 to − 0.03, <italic>P</italic> < 0.05). The inverse association was more consistent among men, non-Hispanic White participants, and those without diabetes or cancer, with significant interactions (all <italic>P</italic> for interaction < 0.01). Serum 25(OH)D3 and insulin each significantly mediated part of the association (both <italic>P</italic> < 0.05). Conclusions Healthier behavioral rhythm profiles were associated with lower biological age acceleration among U.S. adults. Serum 25(OH)D3 and insulin may partly explain this association, suggesting potential metabolic and endocrine pathways. Longitudinal studies are needed to confirm these findings. </p>