Patients with low back pain (LBP) may receive osteopathic manipulative treatment (OMT) to resolve or manage their pain. The indication for OMT for patients with LBP is the presence of somatic dysfunction, diagnosed using palpatory examination. Because palpatory findings commonly have poor interexaminer reliability, the current study used ultrasonography (US) to establish pre-OMT and post-OMT musculoskeletal measurements of relative asymmetry between pelvic and sacral bony landmarks. To document objective musculoskeletal changes that occur in response to OMT using US and to compare palpatory assessment of landmark asymmetry with US assessment. Sixty men and women aged 20 to 55 years with at least 1 episode of LBP in the past 2 weeks were assigned to a seated control, walking control, or OMT group (20 participants per group). Participants received an initial, bilateral US measurement of the skin to posterior superior iliac spine (SPSIS), skin to sacral base position (SBP), and sacral sulcus depth (SSD). Participants in seated control and OMT groups received a palpatory assessment of SBP and SSD prior to initial US assessment. After assessment, the seated control group sat in a waiting room for 30 minutes, the walking control group walked for 5 minutes, and the OMT group received OMT to address sacral base asymmetry using predominantly direct techniques for a maximum of 20 minutes. Participants then received a second US assessment of the same structures. Body mass index (BMI) was correlated with SPSIS (r=0.5, P=.001) and SBP (r=0.6, P<.001). More participants in seated control (75%) and OMT (65%) groups had an increase in asymmetry from first to second US assessment for SPSIS compared with participants in the walking control group (35%, P=.05). No significant differences were found between groups for absolute asymmetry or total change in asymmetry (all P>.10). The κ was -0.1 (95% CI, -0.2 to 0.03) for SBP and -0.01 (95% CI, -0.1 to 0.1) for SSD. Musculoskeletal changes in SPSIS and SBP measurements related to OMT could not be readily identified using US. The SPSIS and SBP measurements were dependent on BMI, which may have affected the accuracy of US to detect small changes in asymmetry. Qualitative palpatory assessments did not correlate with US measurements. Further study is needed to identify US measurements that demonstrate change with OMT. (ClinicalTrials.gov number NCT02820701).