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Although diaphragmatic motion is readily studied by ultrasonography, the procedure remains poorly codified. The aim of this prospective study was to determine the reference values for diaphragmatic motion as recorded by M-mode ultrasonography.Two hundred ten healthy adult subjects (150 men, 60 women) were investigated. Both sides of the posterior diaphragm were identified, and M-mode was used to display the movement of the anatomical structures. Examinations were performed during quiet breathing, voluntary sniffing, and deep breathing. Diaphragmatic excursions were measured from the M-mode sonographic images. In addition, the reproducibility (inter- and intra-observer) was assessed.Right and left diaphragmatic motions were successfully assessed during quiet breathing in all subjects. During voluntary sniffing, the measurement was always possible on the right side, and in 208 of 210 volunteers, on the left side. During deep breathing, an obscuration of the diaphragm by the descending lung was noted in subjects with marked diaphragmatic excursion. Consequently, right diaphragmatic excursion could be measured in 195 of 210 subjects, and left diaphragmatic excursion in only 45 subjects. Finally, normal values of both diaphragmatic excursions were determined. Since the excursions were larger in men than in women, the gender should be taken into account. The lower limit values were close to 0.9 cm for women and 1 cm for men during quiet breathing, 1.6 cm for women and 1.8 cm for men during voluntary sniffing, and 3.7 cm for women and 4.7 cm for men during deep breathing.We demonstrated that M-mode ultrasonography is a reproducible method for assessing hemidiaphragmatic movement.