Multi‐omic analysis of biological aging biomarkers in long‐term calorie restriction and endurance exercise practitioners: A cross‐sectional study

Calorie restriction (CR) and physical exercise (EX) are well-established interventions known to extend health span and lifespan in animal models. However, their impact on human biological aging remains unclear. With recent advances in omics technologies and biological age (BioAge) metrics, it is now possible to assess the impact of these lifestyle interventions without the need for long-term follow-up. This study compared BioAge biomarkers in 41 middle-aged and older adult long-term CR practitioners, 41 age- and sex-matched endurance athletes (EX), and 35 sedentary controls consuming Western diets (WD), through PhenoAge: a composite score derived from nine blood-biomarkers. Additionally, a subset of participants (12 CR, 11 EX, and 12 WD) underwent multi-omic profiling, including DNA methylation and RNAseq of colon mucosa, blood metabolomics, and stool metagenomics. A group of six young WD subjects (yWD) served as a reference for BioAge calculation using Mahalanobis distance across six omic layers. The results demonstrated consistently lower BioAge biomarkers in both CR and EX groups compared to WD controls across all layers. CR participants exhibited lower BioAge in gut microbiome and blood-derived omics, while EX participants had lower BioAge in colon mucosa-derived epigenetic and transcriptomic markers, suggesting potential tissue-specific effects. Multi-omic pathway enrichment analyses suggested both shared and intervention-specific mechanisms, including oxidative stress and basal transcription as common pathways, with ether lipid metabolism uniquely enriched in CR. Despite limitations due to sample size, these findings contribute to the broader understanding of the potential anti-aging effects of CR and EX, offering promising directions for further research.