OBJECTIVE: To test the reliability of a rehabilitative ultrasound imaging (RUSI) protocol for analyzing lumbar multifidus morphologic characteristics for use by clinicians, and to analyze the specificity of isolated lumbar extension resistance exercise (ILEX) DESIGN: Intrarater reliability and interrater (imaging and quantification) reliability. METHODS: A standardized RUSI protocol was developed and the reliability of the procedure and image analysis (intraclass correlations coefficient [ICC], standard error of the measurement [SEM], minimum detectable change [MDC%]) was analyzed. Lumbar multifidus RUSI measures of muscles thickness, cross-sectional area, and echogenicity were obtained before and after 1 ILEX set with uniform time under tension and range of motion (12°-42°). RESULTS: Intrarater (between-day) reliability was similar (ICC, 0.88-0.97; SEM, 0.14-5.08; MDC%: 11-20) compared to interrater (same day) imaging reliability (ICC, 0.91-0.99; SEM, 0.09-3.78; MDC%, 8-15) and interrater quantification reliability (ICC, 0.83-0.97; SEM, 0.07-2.80; MDC%: 6-25). There was a significant increase of lumbar multifidus muscle thickness (pre-ILEX, 2.94 cm; post-ILEX, 3.16 cm; P<.001) and lumbar multifidus cross-sectional area (pre-ILEX, 7.61 cm2; post-ILEX, 8.42 cm2; P<.001) but a nonsignificant effect on echogenicity (pre-ILEX, 73.12; post-ILEX, 72.05; P = .257). CONCLUSION: RUSI is a reliable and easy-to-use diagnostic tool for lumbar multifidus muscle quality and morphology in daily practice. The tool is sensitive to change as shown using ILEX. JOSPT Open 2024;2(4):354-363. Epub 7 August 2024. doi:10.2519/josptopen.2024.0253