Bile acids (BA) play an important role in lipid metabolism. They facilitate intestinal lipid absorption, and BA synthesis is the main catabolic pathway for cholesterol. The objective of this study was to investigate associations of age, sex, diet (fat intake) and parameters of lipid metabolism (triglycerides, LDL, HDL, body fat content) with fasting plasma BA concentration of healthy individuals. Fasting plasma samples from a cross-sectional study were used to determine the concentrations of 14 BA using an LC-MS stable isotope dilution assay. Triglycerides, LDL and HDL were analyzed by standard clinical chemistry methods and body fat content was measured with a DXA instrument. The dietary fat intake of the 24 h period prior to the sampling was assessed on the basis of a 24 h recall. Subsequent statistical data processing was done by means of a median regression model. Results revealed large inter-individual variations. Overall, higher median plasma concentrations of BA were observed in men than in women. Quantile regression showed significant interactions of selected BA with age and sex, affecting primarily chenodeoxycholic acid and its conjugates. No associations were found for LDL and the amount of fat intake (based on the percentage of energy intake from dietary fat as well as total fat intake). Additional associations regarding body fat content, HDL and triglycerides were found for some secondary BA plasma concentrations. We conclude that age and sex are associated with the fasting plasma concentrations. Those associations are significant and need to be considered in studies investigating the role of BA in the human metabolism.