Background - Lifestyle behaviours such as physical activity, sedentary behaviour and dietary habits have been shown to influence blood lipid levels, and both lifestyle and blood lipids may be associated with haemolysis during storage of blood products. We aimed to investigate whether lifestyle behaviours are associated with degree of haemolysis in red cell concentrates (RCC), and if such associations are mediated by low-density lipoprotein (LDL) cholesterol and triglyceride levels. Materials and methods - Cross-sectional analyses were performed in data from 760 Dutch blood donors participating in Donor InSight, an observational cohort study. Linear regression analyses were conducted to assess associations of lifestyle behaviours with haemolysis levels in RCC 28 days after blood sampling. Lifestyle behaviours included moderate-to-vigorous physical activity and sedentary behaviour measured by accelerometry, and self-reported intake of a selection of foods potentially related to blood lipids, i.e. consumption of eggs, meat, nuts and fish. Potential mediating roles of both LDL cholesterol and triglyceride levels were investigated separately. All analyses were adjusted for relevant confounders. Results - No statistically significant nor substantial associations of any of the lifestyle behaviours with haemolysis in RCC were found, nor were there any associations between lifestyle behaviours and blood lipids. We did find consistent positive associations of LDL cholesterol and triglyceride levels with haemolysis in RCC during storage. Discussion - In this large cohort, blood lipid levels were consistently associated with haemolysis in RCC. Nonetheless, there was no evidence for an association between lifestyle behaviours and haemolysis in RCC, or for mediating effects by blood lipid levels.