To investigate changes in breast density (BD) during the menstrual cycle in young women in comparison to inter-breast and -segment changes as well as reproducibility of a novel Speed-of-Sound (SoS) Ultrasound (US) method.SoS-US uses a conventional US system with a reflector and a software add-on to quantify SoS in the retro-mammillary, inner and outer segments of both breasts. Twenty healthy women (18-40 years) with regular menstrual cycles were scanned twice with two weeks in-between. Three of these were additionally measured twice per week for 25 days. Average SoS (m/s) and ΔSoS (segment-variation SoS; m/s) were measured. Variations between follicular and luteal phases and changes over the four-week period were assessed. Inter-examiner and inter-reader agreements were also evaluated. Variances between cycle phases, examiners and readers were compared.No significant SoS difference was observed between follicular and luteal phases for the twenty women (P = .126), and between all different days for the three more frequently measured women (P = .892). Inter-reader (ICC = 0.999) and inter-examiner (ICC = 0.990) agreements were high. The SoS variance due to menstrual variations was not significantly larger than the inter-examiner uncertainty (P = .461). Inter-reader variations were significantly smaller than menstrual and examiner variations (P < .001).SoS-US showed high inter-examiner and inter-reader reproducibility. The alterations during the menstrual cycles were not significantly larger than the confidence interval of measurements.