Stadiometry measures total trunk height variations but cannot quantify individual spinal segment height changes. Different methods exist to measure both intervertebral disc and lumbar spine height (LSH) variations but they are either limited by radiation exposure or cost. Musculoskeletal ultrasound could be a valuable alternative to measure spinal segmental height changes as a result of intervention. To validate the use of musculoskeletal ultrasound (MSU) and new anatomical landmark references used in assessing inter-mammillary distances (IMD) and LSH changes resulting from lumbar spine traction. Two unembalmed cadaveric lumbar spines were extracted to assess (1) the reliability and validity of MSU, as compared to caliper, for measuring in vitro IMD and LSH using alternative anatomical landmarks than previously reported, and (2) the reliability of MSU for measuring in vitro IMD and lumbar spine height changes recorded during standardized mechanical traction up to 1.20 cm. Intra- and inter-rater reliability of musculoskeletal ultrasound for within and between sessions and for all experimental design, Standard Error ranged from 0.01 to 0.02 and from 0.03 to 0.04 cm for IMDs and LSHs, respectively. Root Mean Square Errors ranged from 1.6 to 6.8% and from 1 to 1.1% for IMDs and LSHs, respectively and mean ICC ranged from 0.98 to 1 for LSH. During traction, mean lumbar spine height measurement change using MSU was 1.15 ± 0.03 cm. Bland and Altman plots demonstrated confidence intervals included in the limits of agreement. Nevertheless, there were significant differences (p< 0.001) for both IMD measurements and lumbar spine height between caliper and ultrasound measurements. Musculoskeletal ultrasound overestimated distances of about 5.5 ± 1.5%. Musculoskeletal ultrasound is reliable and accurate for measuring intersegmental spinal distances and lumbar spine height with an apparent slight overestimation of distances. Based on mean differences, ultrasound technology seems to be valid for measuring lumbar spine height changes and could be suitable for in vivo research.