Abstract Study question Description of uterine contractility characteristics in adenomyosis patients compared to healthy controls and according to degree of dysmenorrhoea measured by quantitative 2D transvaginal ultrasound (TVUS). Summary answer Women with adenomyosis show differences in uterine contraction patterns (frequency, amplitude, velocity, and contraction coordination) compared to healthy controls and depending on degree of dysmenorrhoea. What is known already The natural contraction pattern of the uterus changes throughout the menstrual cycle in response to cyclic changes in hormones. In abnormal uteri, such as adenomyotic uteri, this response may be different. Adenomyosis can cause dysmenorrhoea, menorrhagia, dyspareunia, and subfertility. These symptoms could be explained by different contraction patterns of the uterus, however, this has not yet been objectively quantified due to the absence of a suitable measurement tool. Recently, speckle-tracking and strain analysis of 2D TVUS was used to assess differences in contraction coordination, frequency, velocity, and direction, depending on the phase of the menstrual cycle in healthy women. Study design, size, duration This study is part of an ongoing multi-centre prospective observational cohort study investigating uterine contractility in TVUS recordings. Our study includes the TVUS recordings of 31 women with adenomyosis and 106 women with healthy uteri. Patients were included in 3 centres from 2017 to 2023 (Netherlands, Greece, Italy). Participants/materials, setting, methods 31 women with sonographic suspicion of adenomyosis and 106 women with healthy uteri with natural menstrual cycles were included. Uterine contraction frequency, amplitude, velocity, and coordination were assessed by applying a dedicated speckle tracking and strain analysis to 2-4-minute TVUS recordings in midsagittal section. Degree of dysmenorrhoea was measured according to visual analogue score (VAS). Women with suspicion of adenomyosis were compared to women with healthy uteri according to the phase of their menstrual cycle. Main results and the role of chance Age, BMI, parity and uterus volume were significantly higher in the women with adenomyosis compared to the healthy controls (p < 0.05). Uterine contractility differed between women with adenomyosis and healthy controls during the periovulatory phase, revealing lower frequency (1.50±0.25 vs. 1.75±0.35, p = 0.031), higher amplitude (0.076±0.039 vs. 0.046±0.018, p = 0.001), and lower velocity (0.64±0.18 vs. 0.84±0.22, p = 0.020) of uterine contractions in the group with adenomyosis patients. In the late luteal phase, women with adenomyosis showed higher amplitude (0.050±0.02 vs. 0.035±0.01, p = 0.039) and lower velocity (0.51±0.11 vs. 0.72±0.16, p = 0.032) than the healthy controls. During menstruation, women with adenomyosis showed a trend towards higher contraction amplitude (0.044± 0.01 vs. 0.037±0.01, p = 0.051) compared to healthy controls. During the menstrual, periovulatory and late luteal phase, women with adenomyosis showed reduced contraction coordination (p = 0.047, p = 0.015 and p = 0.011, respectively) compared to healthy controls. Increased dysmenorrhoea (VAS score) in overall adenomyosis patients was associated with lower contraction velocity (p = 0.043) and a tendency towards lower frequency (p = 0.160) and higher amplitude (p = 0.154). Limitations, reasons for caution No sub-analysis was done to assess effects of additional adenomyosis characteristics (i.e. adenomyosis severity, adenomyosis type, adenomyosis location). Women with extensive adenomyosis were not included due to impossibility to perform analysis of ultrasound images. Women with adenomyosis were older, had higher BMI and larger uterus volumes than healthy controls. Wider implications of the findings Uterine contractility differs between patients with adenomyosis versus healthy women throughout the menstrual cycle. More dysmenorrhoea in adenomyosis patients was also associated with a different contractility pattern. This suggests an aetiological mechanism for the clinical presentation of adenomyosis (i.e. dysmenorrhoea and subfertility) and presents potential therapeutic markers. Trial registration number NL52466.100.15