Performance of interferon-gamma release assays (IGRAs) is influenced by preanalytical, laboratory and host factors. The data regarding how critical illnesses influence IGRA results are limited. This study aimed to investigate IGRA performance among critically ill patients. Patients admitted to intensive care unit (ICU) were prospectively enrolled, and underwent QuantiFERON-TB Gold In-Tube testing on admission and discharge. The associations between patient factors and IGRA results were explored. In total, 118 patients were included. IGRA results on admission were positive, negative and indeterminate for 10 (9%), 36 (31%) and 72 (61%) patients. All indeterminate results were due to a low mitogen response. Indeterminate results were associated with higher disease severity and lower serum albumin levels. Ninety (76%) patients survived to ICU discharge and had repeat IGRA testing 13.3 ± 10.1 days after first ones. Of those, 43 (48%) had indeterminate results, and no IGRA conversion or reversion was observed. The majority (35/51, 69%) of ICU survivors with initial indeterminate results still had indeterminates on follow-up testing. Acute critical illnesses exert a significant impact on IGRA performance and a high proportion of indeterminate results was seen in ICU patients. This study highlights limitation of IGRAs in the critically ill and judicious selection of patients to be tested should be considered.