A 12-week hypocaloric Mediterranean diet is associated with metabolic improvement and modulation of diurnal clock gene and innate immune receptor expression in peripheral blood mononuclear cells of patients with obesity

Obesity is characterised by chronic low-grade inflammation and circadian disruption, both of which contribute to metabolic dysfunction. Increasing evidence suggests a bidirectional interaction between molecular clock components and innate immune pathways, particularly Toll-like receptors (TLRs). However, whether dietary interventions can modulate this circadian–immune axis in humans remains poorly understood. This prospective exploratory interventional study investigated the effects of a 12-week Mediterranean Diet (MD) intervention on the expression profile of clock genes and innate immune receptors in peripheral blood mononuclear cells (PBMCs) from patients with obesity. Thirty-eight adults with obesity completed a 12-week MD intervention. Anthropometric, metabolic, dietary, and chronotype assessments were performed at baseline (T0) and after intervention (T1). PBMCs were collected at two time points: 8:00 AM and 4:00 PM. Expression levels of clock genes (BMAL1, PER1, PER2, CRY1, CRY2) and innate immune receptors (TLR4 and TLR8) were quantified by RT-qPCR. Chronotype was evaluated using the Morningness–Eveningness Questionnaire (MEQ). MD was associated with significant reductions in body weight, body mass index, waist circumference, and fat mass percentage, together with increases in fat-free mass, muscle mass, HDL cholesterol, and MEQ score. At baseline, TLR4 and TLR8 expression was significantly higher in the afternoon than in the morning. Following MD, BMAL1 expression significantly increased in the morning, whereas PER1, TLR4, and TLR8 expression significantly decreased in the afternoon. Moreover, BMAL1, PER1, PER2, CRY1, and CRY2 exhibited a more pronounced morning-to-afternoon expression pattern following the intervention, suggesting improved diurnal organization. Significant positive correlations between BMAL1 and both TLR4 and TLR8 were observed at both time points. Although MEQ scores significantly increased, indicating a shift toward greater morningness, no significant changes were observed in chronotype category distribution. A 12-week MD intervention was associated with concomitant improvements in anthropometric status, body composition, chronotype score, and circadian–immune regulation in patients with obesity. Increased morning BMAL1 expression together with reduced afternoon TLR4 and TLR8 expression suggests partial restoration of diurnal–immune homeostasis. These findings support the hypothesis that dietary modulation of the BMAL1–TLR axis may represent a mechanism linking metabolic improvement to diurnal g and identify peripheral clock gene expression as a potential translational biomarker of response to lifestyle interventions. Given the absence of a comparator arm, these findings should be interpreted as associations and require confirmation in randomised controlled studies. ClinicalTrials.gov, NCT06236932, registered 24 January 2024, https://clinicaltrials.gov/study/NCT06236932?tab=study.