We compared the performance of the T-SPOT.TB assay with blood used within 0 to 3.5 h after collection (control) to its performance with blood stored for 0 to 3.5, 5 to 8, 18 to 21, or 31 to 33 h with the addition of T-Cell Xtend (experimental), using samples from 154 participants. The 95.4% concordance between paired specimens indicated that blood can be stored for up to 33 h prior to T-SPOT.TB testing.