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Mammographic breast density has been proposed as a surrogate endpoint in breast cancer prevention studies, but little is known about its variability over time, particularly in relation to menstrual cycle phase. The purpose of this study was to assess variation in breast density on digital mammograms using quantitative and qualitative density measures. Menstrual cycle phase was determined by salivary estradiol and progesterone assays. 73 healthy subjects with regular menses had 1-3 mammograms with paired saliva collection during a 12-month period. The mean difference in density as a percentage of the mean density was calculated for follicular-luteal (n = 50), luteal-luteal (n = 26) and follicular-follicular (n = 23) pairs in the same woman using the same breast. Two density measures (measurement of dense area and BIRADS) were used. The mean luteal density exceeded the mean follicular density by 7.1-9.2%, but density differences between luteal pairs and follicular pairs did not exceed 5%. The intraclass correlation for measurement of dense area was greater than 85% in all phases of the menstrual cycle, but was below 50% for BIRADS for luteal-follicular and follicular-follicular pairs. Our study provides estimates of the amount of variation in mammographic density during the menstrual cycle, and that inherent in repeated density measurement in premenopausal women, and suggests that menstrual phase of mammographic evaluation should be controlled for in intervention studies where density is being used as a surrogate measure.