Introduction This case series aims to compare changes in spinal column mobility using the Spinal Mouse® (Idiag, Volketswil, Switzerland), a device providing a spatial survey of a single vertebra's position. The measurements have been made before and after an osteopathic treatment with different spinal column positions on healthy subjects. We presumed that osteopathic treatment is able to improve spinal column mobility. Methods The measurement was carried out with the naked spinal column in the following positions: standing, static bending, dynamic bending, and conducting the static Matthiass test, which consists of maintaining the standing position with outstretched arms, with a 1 kg weight. We evaluated the vertebral tilt degrees and changes in height (expressed in mm) in the mentioned positions before and after the osteopathic treatment. Results We observed improvements in spinal column tilt (expressed in degrees) and in vertebral metameres length (expressed in mm) in a standing position (T<0.04 and T<0.04). We also noted a global increase in tilt (expressed in degrees) during the static bending position (T<0.05) and in the thoracic tract during the Matthiass test (T<0.02). Conclusions The present study highlighted that osteopathic treatment was able to increase vertebral mobility, concerning tilt (expressed in degrees) and length (expressed in mm), evaluated in different positions.