Endogenous pituitary hormones are commonly used in clinical and epidemiologic studies and some of them are thought to influence the risk of several diseases in women. In most studies, endogenous levels of pituitary hormones are usually assessed at a single point in time, assuming that this single measurement represents the long-term biomarker status of the individual. Such an assumption is rarely tested and may not always be valid. This study examined the reproducibility of the following pituitary hormones: adrenocorticotropic hormone (ACTH), growth hormone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and prolactin, measured using the Luminex xMap method in sera of healthy premenopausal and postmenopausal women. The study included 30 premenopausal women with three yearly samples and 35 postmenopausal women with two repeated yearly samples randomly selected from an existing prospective cohort. Analysis of intraclass correlation coefficients suggested higher reproducibility in postmenopausal women compared with premenopausal women for the following hormones: FSH (0.72 and 0.37, respectively), LH (0.83 and 0.44, respectively), and growth hormone (0.60 and 0.35, respectively). The intraclass correlation coefficients were relatively high and similar between postmenopausal and premenopausal women for ACTH (0.95 and 0.94, respectively), TSH (0.85 and 0.85, respectively), and prolactin (0.72 and 0.69, respectively). This study found that serum concentrations of FSH, LH, and growth hormone are stable in postmenopausal women and that ACTH, TSH, and prolactin are stable in both premenopausal and postmenopausal women, suggesting that a single measurement may reliably categorize average levels over at least a 2-year period.