Background: In ALS patients, diaphragmatic dysfunction is usually assessed by pulmonary function testing (PFTs) that requires patient cooperation. Evidence suggest that diaphragm ultrasound (DU) can be utilized, as an alternative to PFTs, to detect reduced diaphragmatic motility in ALS patients. This study aimed to verify the agreement between the results obtained by DU with those obtained by PFTs in both standing-up and supine positions. Methods: Twenty nine spinal ALS and thirteen healthy controls were studied in standing up and lying 30° supine position. All subjects performed PFTs and DU, to assess of diaphragmatic excursion, delta-thickness between end inspiration and end expiration (ΔT), and the thickening fraction (TF).