AIM: To investigate the accuracy of two semi-automatic segmentation measurements based on magnetic resonance imaging (MRI) three-dimensional (3D) Cube fast spin echo (FSE)-flex sequence in phantoms, and to evaluate the feasibility of determining the volumetric alterations of orbital fat (OF) and total extraocular muscles (TEM) in patients with thyroid-associated ophthalmopathy (TAO) by semi-automatic segmentation. MATERIALS AND METHODS: Forty-four fatty (n=22) and lean (n=22) phantoms were scanned by using Cube FSE-flex sequence with a 3 T MRI system. Their volumes were measured by manual segmentation (MS) and two semi-automatic segmentation algorithms (regional growing [RG], multi-dimensional threshold [MDT]). Pearson correlation and Bland-Altman analysis were used to evaluate the measuring accuracy of MS, RG, and MDT in phantoms as compared with the true volume. Then, OF and TEM volumes of 15 TAO patients and 15 normal controls were measured using MDT. Paired-sample t-tests were used to compare the volumes and volume ratios of different orbital tissues between TAO patients and controls. RESULTS: Each segmentation (MS RG, MDT) has a significant correlation (p<0.01) with true volume. There was a minimal bias for MS, and a stronger agreement between MDT and the true volume than RG and the true volume both in fatty and lean phantoms. The reproducibility of Cube FSE-flex determined MDT was adequate. The volumetric ratios of OF/globe (p<0.01), TEM/globe (p<0.01), whole orbit/globe (p<0.01) and bone orbit/globe (p<0.01) were significantly greater in TAO patients than those in healthy controls. CONCLUSION: MRI Cube FSE-flex determined MDT is a relatively accurate semi-automatic segmentation that can be used to evaluate OF and TEM volumes in clinic. (C) 2018 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.