The purpose of this study is to determine if there are differences in the expression of estrogen-regulated genes (ERGs), proliferation-associated genes and the progesterone effector RANKL, in premenopausal ER+ breast cancer as a result of the major changes in hormone levels that occur through the menstrual cycle. Primary ER+ tumours from 174 patients were assigned to one of three menstrual cycle windows: W1 (days 27-35 + 1-6), W2 (days 7-16) and W3 (days 17-26). RNA expression of 42 genes, including 24 putative genes associated with plasma E2 levels, seven proliferation genes and RANKL was measured. Expression of PGR, TFF1, GREB1 and PDZK1 followed the previously reported pattern: a higher level in W2 compared to W1 while W3 had an intermediate value, mirroring changes in plasma estradiol. Of the other 20 ERGs, four (RUNX1, AGR2, SERPINA3 and SERPINA5) showed significant differences (p = 0.009-0.049) in expression across the menstrual cycle. The expression of six of seven proliferation-associated genes varied across the cycle but differently from the ERGs, being 20-35 % lower in W3 compared to W1 and W2 (p = 0.004-0.031). Expression of RANKL was 2.5 to 3-fold highest in W3 (p = 0.0001) and negatively correlated to the expression of the proliferation-associated genes (r = -0.37; p < 0.0001). Expression of proliferation-associated genes and RANKL in ER+ breast tumours varies across the menstrual cycle showing a different rhythm to that of ERGs. This may affect the interpretation of gene expression profiles but may be exploitable as an endogenous test of endocrine responsiveness.