Background: The underlying causes of indeterminate (invalid) interferon-gamma release assays are not clear and predictors of indeterminate results do not exist. To date the effect of steroid concomitant therapy on the rate of indeterminate tests is unknown. Methods: We performed a retrospective analysis of the results of 1339 tests, including clinical data from patient records, of which 914 were suitable for complete data analysis.Results: 130 (15.2 %) tests were positive and 719 (83.8 %) were negative, 9 tests (1 %) were borderline positive (according to manufacturer’s definition). Indeterminate results (56 (6.1 %) arose from failure of the negative control in 24 (42.9 %) and failure of the positive control in 32 (57.1 %) tests. Of 914 patients, 196 were taking cortisone at the time of testing; among these patients 180 (91.8 %) returned a valid result and 16 (8.2 %) an indeterminate result compared to 40 indeterminate tests of 718 (5.6 %) results from patients not on corticosteroid therapy at the time of testing (p = 0.180), (relative risk [RR] 1.46, [95 % CI 0.84 to 2.56]). No significant difference in the rate of indeterminate results was found with immunosuppressive diseases or medications other than with HIV; among HIV-positive patients, 11 (11.7 %) had an indeterminate test result, compared to 45 (5.5 %) in HIV-negative individuals (p =0.017, relative risk [RR] 2.13, [95 % CI 1.14 to 3.98]).Conclusions: steroid therapy does not have an effect whereas HIV positivity has a negative effect on the number of indeterminate results in the T-Spot.TB test.