Aim of this work was to evaluate the reproducibility of hormone driven regional grey matter volume differences in women, and their correlations with premenstrual symptoms, as determined by voxel-based morphometry (VBM). After data quality control, a total of 138 T1-weighted MR images were included in this longitudinal study, and were analyzed as three different subgroups. Women with a natural menstrual cycle were scanned at three time-points: follicular, ovulatory and luteal phase. Two groups of women, using androgenic and anti-androgenic hormonal contraceptives, respectively, were scanned twice: during the pill-free week and during pill intake. Additionally, subjects were asked to complete a “daily rating of severity of problems” questionnaire, to quantify premenstrual symptoms. All data were analyzed using SPM8 and SPM12 with identical parameter settings. In the natural menstrual cycle group, the regional grey matter volume of the insula is larger at ovulation, as compared to the luteal phase. Premenstrual symptoms correlate differently with regional grey matter volumes between women with a natural cycle and hormonal contraceptive users. Changes in hormonal environment can to various extents affect VBM findings in women. We suggest that researchers take these confounding factors into account while applying this technique, to avoid heterogeneity in data acquisition and to safeguard the sensitivity of findings. Additionally, we suggest validating the consistency of results using more than one software package.