Little is known about the mechanical properties of pelvic floor structures and their role in the course and treatment of pelvic organ prolapse (POP). We hypothesize that in vivo mechanical properties of the vaginal wall are related to the appearance of POP and pre-and post-operative states. We used a suction device for intravaginal application, the aspiration device, to evaluate two in vivo mechanical parameters of the anterior vaginal wall, the load dependent tissue displacement and the initial displacement, by image analysis in pre- and post-menopausal women with (POP) and without (control) cystocele (POP: pre-menopausal: N = 6, post-menopausal: N = 19, control: pre-menopausal: N = 17, post-menopausal: N = 6). Mechanical parameters in women with and without cystocele and pre- and post-operative parameters were compared. Statistically significant differences were observed between the two mechanical parameters in pre- and post-operative states (P = 0.04, P = 0.03), but not between the parameters for women with and without cystocele (P = 0.92, P = 0.75). The mechanical behavior of pelvic floor structures is influenced by factors such as POP, age or estrogenization that are apparent at different length scales, which cannot be separated by the aspiration based biomechanical measurements. When comparing pre- and post-operative states of the same patient, a firmer tissue response was observed after intervention.