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Phytosterols are plant sterols that are structurally similar to cholesterol and are characterized by anti-carcinogenic and anti-atherogenic properties. β-Sitosterol and campesterol are the predominant phytosterols in blood. The present study aimed to analyse the reproducibility and overtime reliability of plasma β-sitosterol and campesterol measurements. In order to study the reproducibility of the measurement (technical variability), three healthy premenopausal women donated a sample of their blood. Each blood sample was subdivided into six aliquots and analysed within the same run by the same laboratory technician. The intraclass correlation coefficients (ICCs) of the assay for plasma β-sitosterol and campesterol were 0.88 and 0.94 (95% confidence intervals low bounds (95% CIlow) were 0.66 and 0.82), respectively. To study the reliability of β-sitosterol and campesterol measurement over time, seven premenopausal women were recruited. Over a 6-month period, each woman provided a fasting blood sample once a month at the same time of day, and the same numerical day of the luteal phase of her menstrual cycle (between the 20th and 24th day of her menstrual cycle). All plasma samples from the same individual were processed together at the same time by the same technician at the end of the 6-month period. The overtime ICCs of plasma β-sitosterol and campesterol were 0.91 (95% CIlow 0.49) and 0.58 (95% CIlow 0.31), respectively. The high reproducibility and good overtime reliability of plasma β-sitosterol and campesterol measurements indicate that they may be suitable for potential clinical and population-based studies on cancer prevention.