AbstractAnimal sterols, plant sterols and bile acids in stool samples have been suggested as biomarkers of dietary intake. It is still unknown whether they also reflect long-term habitual dietary intake and can be used in aetiological research. In a subgroup of the Cooperative Health Research in the Augsburg Region (KORA FF4) study, habitual dietary intake was estimated based on repeated 24-h food list and a FFQ. Stool samples were collected according to a standard operating procedure and those meeting the quality criteria were extracted and analysed by means of a metabolomics technique. The present study is based on data from 513 men and 495 women with a mean age of 60 and 58 years, respectively, for which faecal animal and plant sterols and bile acids concentrations and dietary intake data were available. In adjusted regression models, the associations between food intake and log-normalised metabolite concentrations were analysed. Bonferroni correction was used to account for multiple testing. In this population-based sample, associations between habitual dietary intake and faecal concentrations of animal sterols were identified, while the impact of usual diet on bile acids was limited. A habitual diet high in ‘fruits’ and ‘nuts and seeds’ is associated with lower animal faecal sterols concentrations, whereas a diet high in ‘meat and meat products’ is positively related to faecal concentrations of animal sterols. A positive association between glycocholate and fruit consumption was found. Further studies are necessary for evaluation of faecal animal sterols as biomarkers of diet. The findings need to be confirmed in other populations with diverse dietary habits.