The strategic use of the gamma-interferon (IFN-gamma) assay (Bovigam) can provide a means for the early identification of Mycobacterium bovis infected cattle, thus ensuring their removal from an infected herd. It has been reported that performance of the test can be influenced by various factors including a recent tuberculin skin test and the length of delay between collection and processing of blood samples. In this study, single intradermal comparative tuberculin test (SICTT) reactor and non-reactor cattle were recruited from herds infected with M. bovis and grouped according to their SICTT responses. Group 1 comprised reactor cattle selected on the basis of their SICTT response to PPD-bovine (purified protein derivative of tuberculin) exceeding that of PPD-avian by at least 12mm. Group 2 animals were selected from herds undergoing routine surveillance for bovine tuberculosis and contained standard SICTT reactor cattle (PPD-bovine exceeding that of PPD-avian by at least 4mm) and non-reactors. We investigated the effects of the SICTT on the assay results by measuring the in vitro IFN-gamma responses of Group 1 reactor cattle at time intervals pre- and post-skin test. No significant differences were measured in the IFN-gamma responses of the reactor animals to PPD-bovine and PPD-avian for up to 65 days. To investigate if a delay in processing of blood affected the performance of the assay, we compared results using duplicate blood samples from Group 1 and Group 2 cattle stimulated with PPD antigen at 8h and at 24h after collection. In both groups of animals the mean optical density (OD) values of the assay at 24h post-collection were significantly lower than those at 8h. Our results demonstrated that a delay in processing of the blood samples from cattle subjected to routine surveillance could significantly impact on the outcome of the IFN-gamma assay resulting in a change of the IFN-gamma status of the animals.