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Background: The European Society of Cardiology recommends that patients with greater than 10% area of ischemia should receive revascularization. We investigated inter-observer variability for the extent of ischemic defects reported by different physicians and by different software tools, and if inter-observer variability was reduced when the physicians were provided with a computerized suggestion of the defects. Methods: Twenty-five myocardial perfusion single photon emission computed tomography (SPECT) patients who were regarded as ischemic according to the final report were included. Eleven physicians in nuclear medicine delineated the extent of the ischemic defects. After at least two weeks, they delineated the defects again, and were this time provided a suggestion of the defect delineation by EXINI Heart(TM) (EXINI). Summed difference scores and ischemic extent values were obtained from four software programs. Results: The median extent values obtained from the 11 physicians varied between 8% and 34%, and between 9% and 16% for the software programs. For all 25 patients, mean extent obtained from EXINI was 17.0% (+/- standard deviation (SD) 14.6%). Mean extent for physicians was 22.6% (+/- 15.6%) for the first delineation and 19.1% (+/- 14.9%) for the evaluation where they were provided computerized suggestion. Intra-class correlation (ICC) increased from 0.56 (95% confidence interval (CI) 0.41-0.72) to 0.81 (95% CI 0.71-0.90) between the first and the second delineation, and SD between physicians were 7.8 (first) and 5.9 (second delineation). Conclusions: There was large variability in the estimated ischemic defect size obtained both from different physicians and from different software packages. When the physicians were provided with a suggested delineation, the inter-observer variability decreased significantly.