A Randomized Crossover-Controlled Trial Investigating the Epigenetic Impact of a Greens-Based Supplement in Adults

Abstract

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. However, limited research has focused on greens-based supplements and their impact on epigenetic markers of aging and metabolic health. This 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 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. A significant treatment effect was observed for the Horvath clock (p = 0.015), with an unexpected increase in epigenetic age during supplementation. No significant changes were observed in AdaptAge, PCGrimAge, or DamAge. 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 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 microbial taxa, without significant effects on overall microbiome diversity, metabolic health markers, or body composition. Exploratory correlations suggested links between microbial diversity, glycemic control, and specific taxa with epigenetic clocks. These findings highlight the value of using multiple epigenetic clocks in nutritional studies, given the varied responses observed across different clocks.