To assess whether with recent MR technology current patient preparation literature recommendations for female pelvic MRI are still valid and how they are influencing the position of the female pelvic organs, image quality, and diagnostic confidence. This prospective study was performed in two centres. The effects of bladder filling (empty, moderate, full; n = 26), fasting, saturation band (n = 25), and menstrual cycle (n = 25) were assessed in healthy subjects in centre 1, while the effect of intravenous glucagon application was evaluated in 20 patients with benign conditions in centre 2. Images of the pelvis were acquired using 1.5-T MRI with T2-weighted turbo spin echo imaging in sagittal and (angulated) transaxial planes. The analysis was conducted hierarchically using paired Wilcoxon tests with an alpha significance level of 0.05. Urinary bladder filling influenced the cervix-to-uterine angle (p < 0.001) but had no clear effect on image quality (p > 0.05). A moderately full bladder provided the best delineation of fat between the bladder and uterus (p = 0.0009). A full bladder resulted in highest ovarian displacement (p = 0.0059). Timing within the menstrual cycle did not influence the depiction of zonal anatomy (p > 0.05). Fasting (p < 0.02) and saturation bands (p < 0.001) had a positive effect on image quality. Glucagon was associated with decreased MRI artefacts (p = 0.002). Fasting, antispasmodic agents, and saturation bands improved MR image quality and are recommended for female pelvic MRI. Urinary bladder filling influenced the cervix-to-uterine angle, but had no clear effect on image quality. • Fasting, saturation band, and glucagon injection significantly reduced bowel movement artefacts. • Depiction of the anatomical details of the uterus was not influenced by the timing of MRI within the menstrual cycle. • Although bladder filling did not impact diagnostic capabilities in healthy volunteers, in clinical practice, it may be useful to examine patients with a moderately full bladder, to reduce diagnostic inaccuracies.