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Background and rationale. Epidemiologic research suggests that physical activity (PA) reduces the risk of chronic diseases including gallstones. Objective. This study explores the association between recreational physical activity (RPA) and risk of asymptomatic gallstones (AG) in adult Mexican women.Material and methods. We performed a cross-sectional analysis of women from the Health Workers Cohort Study. The study population included Mexican women aged 17-94 years, with no history of gallstone (GS) or cholecystectomy. A self-administered questionnaire was used to collect information on weight change, gynecological health history, cholesterol-lowering medications and diuretics, history of diabetes mellitus type 2 (DM2), PA and diet. PA was calculated in minutes/day, minutes/week and Metabolic Equivalents (METs)/week. Gallstone diagnosis was performed using real-time ultrasonography. The association between RPA and risk of AG was evaluated using multivariate logistic regression models.Results. Of the 4,953 women involved in the study, 12.3% were diagnosed with AG. The participants with AG were significantly older, had a higher body mass index, and had a higher prevalence of DM2 than those without AG. The participants with > 30 min/day of RPA had lower odds of AG (OR = 0.80; 95% CI: 0.65-0.97; P = 0.03), regardless of other known risk factors for gallstone disease. Furthermore, we observed an inverse relationship between RPA time and AG risk, especially in women doing more than 150 min a week of RPA (OR = 0.76; 95%CI: 0.61-0.95; P = 0.02).Conclusion. These findings support the hypothesis that RPA may protect against AG, although further prospective investigations are needed to confirm this association.