Objective: The purpose of this study was to investigate the effect of a session of osteopathic manipulative techniques on diaphragmatic motion and thickness in healthy participants. Methods: This was a prospective, randomized, double-blinded, case vs sham vs control clinical trial performed in an outpatient osteopathic clinic in Rome, Italy. Sixty-seven healthy participants, mean age 40.4 ± 14.5 years, received an ultrasound evaluation of diaphragmatic motion and thickness, followed by a systematic osteopathic evaluation. After randomization, the experimental group (n = 22) received osteopathic manipulation, whereas the sham (n = 22) and the control (n = 22) groups had a light touch approach and simple observation, respectively. After a 1-session intervention, new osteopathic and ultrasound assessments were repeated in all participants. Results: A statistically significant increase in diaphragmatic mobility was observed in the experimental group after the osteopathic manipulation (Δ = 14.5 mm, P b .001; analysis of variance P b .001 vs both sham: Δ = -0.22 mm, and control: Δ = -2.09 mm groups). A strong linear relationship was observed between the diaphragmatic motion gradient, measured with ultrasonography, and the score assigned by the operator evaluating the change of diaphragm mobility after intervention. Conclusion: Osteopathic techniques used in this study improved the diaphragmatic motion (but not the muscle thickness) in healthy participants. Further studies are needed to confirm our findings and eventually identify the clinical conditions that may benefit from osteopathic manipulative treatment of the diaphragm. (J Manipulative Physiol Ther 2019;42:47-54)