A method was developed using 3D stress magnetic resonance imaging (MRI) and was piloted to test hypotheses concerning changes in apical ligament lengths and lines of action from rest to maximal Valsalva. Ten women with (cases) and ten without (controls) pelvic organ prolapse (POP) were selected from an ongoing case-control study. Supine, multiplanar stress MRI was performed at rest and at maximal Valsalva and was imported into 3D Slicer v. 3.4.1 and aligned. The 3D reconstructions of the uterus and vagina, cardinal ligament (CL), deep uterosacral ligament (USL(d)), and pelvic bones were created. Ligament length and orientation were then measured. Adequate ligament representations were possible in all 20 study participants. When cases were compared with controls, the curve length of the CL at rest was 71 ±16 mm vs. 59 ± 9 mm (p = 0.051), and the USL(d)was 38 ± 16 mm vs. 36 ± 11 mm (p = 0.797). Similarly, the increase in CL length from rest to strain was 30 ± 16 mm vs. 15 ± 9 mm (p = 0.033), and USL(d) was 15 ± 12 mm vs. 7 ± 4 mm (p = 0.094). Likewise, the change in USL(d) angle was significantly different from CL (p < 0.001). This technique allows quantification of 3D geometry at rest and at strain. In our pilot sample, at maximal Valsalva, CL elongation was greater in cases than controls, whereas USL(d) was not; CL also exhibited greater changes in ligament length, and USL(d) exhibited greater changes in ligament inclination angle.