Table 1_Epigenetic and microbiome responses to greens supplementation in obese older adults: results from a randomized crossover-controlled trial.docx

Aging is influenced by genetic, environmental, and lifestyle factors. Preliminary studies suggest that fruit and vegetable-based dietary supplements may reduce inflammation and oxidative stress, key factors in aging. Greens-based supplements typically contain concentrated extracts of leafy greens, fruits, vegetables, and bioactive phytochemicals, providing micronutrients and polyphenols that may influence aging-related pathways. This exploratory study evaluated the effects of a 30-day greens-based supplement on epigenetic markers of aging and metabolic health in adults aged 50–65 years with body mass index (BMI) >30 kg/m2, using a 60-day randomized crossover design. Participants were randomized to immediate or delayed supplementation. During the 30-day intervention period, participants consumed a daily greens supplement. Primary outcomes included peripheral blood mononuclear cell DNA methylation and epigenetic age (Horvath, PCGrimAge, AdaptAge, and DamAge). Secondary measures included clinical metabolic biomarkers, microbiome diversity, breath hydrogen and methane, body composition, actigraphy, dietary intake, and quality of life questionnaires [RAND 12 item short form questionnaire (SF-12), and 21-item Depression, Anxiety, and Stress Scale (DASS-21)]. Twenty-one participants began the protocol (65% female, mean age 58.4 ± 5.3 years, mean BMI 38.1 ± 8 kg/m2). Nineteen participants completed the study. Horvath clock data indicated that biological age paradoxically increased during the supplementation period, whereas newer-generation clocks (AdaptAge, DamAge) demonstrated trends toward improved outcomes. Gut microbiome alpha diversity remained stable; taxa of interest, including Bilophila (p = 0.037) and Desulfobacterota (p = 0.031) changed with supplementation. Body composition, metabolic biomarkers, dietary intake, breath gases, sleep, and psychosocial measures were unchanged during the study. Exploratory pre-to-post supplementation change score correlations found no significant associations between epigenetic clocks and secondary outcomes, except for an inverse relationship between Faith's phylogenetic diversity and fasting blood glucose (rs = −0.81, p < 0.001). In summary, 30 days of greens-based supplementation led to selective changes in epigenetic aging markers and individual gut microbial taxa, without significant effects on overall microbiome diversity, metabolic health markers, or body composition. Additionally, exploratory correlations suggest potential links between changes in microbial diversity and glycemic control following greens supplementation.