Abstract
<title>Abstract</title> <p> Background Ultra-processed foods (UPF) have been increasingly linked to adverse health outcomes, yet the biological pathways underlying this association remain poorly understood. Epigenetic mechanisms regulate system homeostasis and are highly responsive to environmental influences, including dietary exposures. Objective To identify a reproducible and modifiable DNA methylation fingerprint associated with UPF intake across different study designs. Methods The study integrated data from three Spanish cohorts: ENRICA-Seniors II, using an extreme phenotype case–control design that selected 50 participants with the lowest and 50 with the highest energy-adjusted UPF intake; PREDIMED-Plus, a longitudinal analysis of individuals who sustained reductions in UPF intake over 6 months (n = 18); and CORDIOPREV, a postprandial challenge study (0–4 h) conducted in patients with coronary heart disease (n = 129). DNA methylation was profiled using the Infinium MethylationEPIC BeadChip and analyzed with <italic>limma</italic> mixed-linear models, adjusting for age, sex, smoking, alcohol, BMI/weight and estimated blood cell proportions. Pathway enrichment analyses were used to interpret the biological relevance of identified DMPs. Results A total of 1,192 DMPs were identified in ENRICA-Seniors II, 2,666 in PREDIMED-Plus, and 2,602 in CORDIOPREV (FDR < 0.05). Cross-cohort integration revealed 32 annotated genes consistently overlapping across the three study designs, primarily involved in endothelial signalling and angiogenesis, DNA-damage response and autophagy, neuronal and synaptic function, and metabolic regulation. Conclusions This study provides evidence that UPF consumption may be associated with a reproducible and modifiable DNA methylation signature across heterogeneous study designs, involving genesin key physiological pathways. While causal inference cannot be established, these findings underscore the need for further research to elucidate the role of UPF-related epigenetic alterations in disease development. </p>