Background: Blood biomarkers of atrial fibrillation (AF)-related pathways can characterize the atrial substrate promoting the development of AF. Exploring associations of blood biomarkers with left atrial (LA) structure and function could enhance our understanding of AF etiopathogenesis and inform AF prevention strategies. Methods: We studied 532 participants of the PREDIMED-Plus trial, a multicenter randomized trial in overweight and obese adults (55-75 years) with the metabolic syndrome in Spain. The trial objective was to test a lifestyle intervention for primary prevention of cardiovascular disease. At baseline, 3 and 5 years after randomization, these participants underwent transthoracic echocardiography and provided blood for serum biomarker measurements [propeptide of procollagen type I (PICP), high-sensitivity (hs) troponin T (hsTnT), hs C-reactive protein (CRP), 3-nitrotyrosine (3-NT), and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP)]. Outcomes of interest included LA peak systolic longitudinal strain (LA PSLS), LA volume index (LAVi), LA function index (LAFi), and LA stiffness index (LASi). We performed cross-sectional and longitudinal analyses to evaluate relationships between log-transformed biomarkers and echocardiographic measurements using multiple linear regression and mixed models: 1) baseline biomarkers and LA outcomes; 2) baseline biomarkers and change in LA outcomes from baseline through year 5; 3) concomitant changes in biomarkers and LA outcomes from baseline to year 5. Results: The participants in this analysis had a mean age of 65 (SD 4.8) years, 40% females, and BMI of 32.2 (SD 3.3) kg/m 2 . At baseline, increased NT-proBNP was associated with worse LAVi (0.78 mL/m 2 , 95% CI 0.04, 1.52), LAFi (-3.61, 95% CI -6.75, -0.46), and LASi (0.02, 95% CI 0.00, 0.04). Higher baseline NT-proBNP was associated with a greater decrease in LA PSLS at year 3 (-0.82%, 95% CI -1.57, -0.06) but not year 5 (-0.01%, 95% CI -0.77, 0.75), and an increase in LASi at years 3 (0.04, 95% CI 0.01, 0.06) and 5 (0.02, 95% CI 0.00, 0.04). Over 5 years, larger elevation in NT-proBNP was associated with more decline in LA PSLS (-0.19%, 95% CI -0.35, -0.02) and more elevation in LAVi (0.28 mL/m 2 , 95% CI 0.10, 0.45) each year. PICP, hsTnT, CRP and 3-NT did not show consistently significant associations with LA outcomes at baseline and through 5 years. Conclusion: In an overweight and obese population, higher NT-proBNP was associated with LA volume enlargement and worsening LA function over 5 years. The implications of these findings for prevention and prediction of AF warrant further investigation.