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Background: Assessing diaphragm mobility is important to detect malfunctions or impending exhaustion and to evaluate the effects of many chest and abdominal conditions on respiratory mechanics. Amongst several imaging methods, ultrasonography represents the only noninvasive, nonionizing imaging technique widely available for the direct assessment of diaphragm excursion. Objectives: the aim of this study is to prospectively assess the supine diaphragmatic motion amplitude, intra- and inter-observer agreement, and anthropometric correlates of diaphragm motion variability, measured through M-mode ultrasonography, in a sample of healthy volunteers. Methods: One-hundred healthy volunteers were considered eligible for the study. Instead of B-mode imaging, the M-mode technique was used to achieve a more accurate measurement of diaphragm motion. To assess intra-observer variability, 3 consecutive measurements (M-mode and B-mode) of the right dome motion were obtained at every session. To test for inter-rater reliability, the subjects were asked to provide 2 more diaphragm motion measurements every week, each performed by 2 experienced operators, and 42 subjects accepted. Results: Diaphragmatic motion was positively correlated with height and weight both at quiet (Spearman’s coefficient = 0.514, p < 0.001 and 0.314, p = 0.038) and deep breathing (Spearman’s coefficient = 0.342, p = 0.001 and 0.225, p = 0.024, respectively) and negatively correlated with age, but only during deep breathing (Spearman’s coefficient = –0.272, p = 0.006). Intra-observer agreement degree on all 3 measurements was excellent during both quiet and deep breathing, with a Cronbach’s alpha of 0.793 and 0.901, respectively, and an intra-class coefficient of 0.797 and 0.900, respectively. Similarly, the degree of inter-observer agreement achieved a Cronbach’s alpha of 0.638 and 0.776, and an inter-class coefficient of 0.632 and 0.778, respectively. Deep breathing was associated with sex only in linear multivariable models (B = –10.14; 95% confidence interval [CI] –17.86, –2.41; p = 0.011), while quiet breathing resulted to be affected by height only (B = 30.05; 95% CI 0.79–59.31; p = 0.044). Conclusions: Diaphragm excursion measurements using the M-mode technique were accurate and could be reproduced also when obtained in recumbent patients. After adjustments, the main predictors of diaphragmatic motion were sex and height, which should be considered to design a specifically tailored study and to develop normality reference equations.