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Purpose: We have focused on the two-dimensional (B-mode) and the time-motion (M-mode) analysis of the zone of apposition to determine the reliability of diaphragm ultrasonography in the clinical environment., Methods: Ten healthy volunteers were enrolled and studied by three operators with different skills in ultrasonography. For every volunteer, each operator acquired three images of the diaphragm for each side, both in B-mode and in M-mode. Then a fourth operator calculated the thickening fraction (TF), by means of the formula TF = (TEI - TEE)/TEE (TEI is the thickness at end inspiration and TEE the thickness at end expiration). Afterwards, intraclass correlation coefficients (ICCs) were computed on TF to establish reproducibility and repeatability both in the B- and M-modes. A Coefficient of Repeatability or repeatability (CR) ≤ 0.3 was considered acceptable., Results: Both B-mode (CRs 0.16-0.26) and M-mode (CRs 0.10-0.15) were sufficiently repeatable to assess TF, except for the less experienced operator (CRs B-Mode 0.20-0.32). Reproducibility was moderate to good between operators with CRs much narrower for the M-Mode (0.13-0.14)., Conclusions: The results of our study have shown that diaphragm ultrasound is repeatable and reproducible when carried out by a radiologist or an intensivist with a basic curriculum in ultrasonography. The method is more accurate when using the M-mode for less experienced operators, and in this case, repeatability and reproducibility are not sufficient to make clinical decisions. No TF value lower than 36% was obtained using both techniques. This suggests the existence of a cut-off value that could be used as an initial tool to discriminate healthy subjects from those affected by diaphragmatic dysfunction., Clinical Trial Registration: EUDRACT 2015-004635-12., (© 2020. Società Italiana di Ultrasonologia in Medicina e Biologia (SIUMB).)