Variability of bone marker measurements is a major problem in their clinical application. Most studies on marker variability have been performed in healthy subjects and over relatively short intervals of time. We prospectively evaluated the long-term variability of bone markers in 102 postmenopausal women diagnosed with primary breast cancer. During follow up (8-48, median 30 mo.), no patient developed bone metastases or other skeletal disease. Patients were seen every 3 months and exactly timed blood/urine specimens were obtained. All analyses were performed after study end by the same technician, using a single batch of reagents per analyte. The coefficient of variation was calculated as CV (%) = square root(sigma(CVi2)/n) (CVi = SD/mean x 100; n = n of CVi). The least significant change (LSC) was then LSC (%) = Z x CV x square root(2). Z = 1.96 for a 95% confidence interval (LSC-95). In a subset of n = 10 patients with no potential interference during follow-up, lowest CVs were recorded for serum (s) calcium (5%), sTAP (12%) and sBAP (14%). The LSC-95 for these markers were 14%, 33% and 39%, respectively. Highest CVs were seen with urine (56%) and serum (42%) CTX (LSC-95: 155%, 117% resp.). We conclude that in breast cancer patients without bone metastases, long-term variability varied greatly between markers. For certain markers, the LSC seems considerably higher than previously reported.