The Bovigam® assay (CSL, Australia) detects IFN-γ released in response to M. paratuberculosis antigen in a whole blood assay. While there are published data on optimisation of the assay for cattle, there is little information for sheep. Investigations were conducted in sheep on anticoagulant, sample transit temperature and time delay before incubation and duration of incubation. Blood from vaccinated (Gudair®) Merino sheep was collected into tubes containing different anticoagulants and incubated at 37 ºC for 24, 48 or 72 hours in either media alone or media containing M. ptb. antigen. Plates containing blood and media ± M. ptb. antigen (added either at plate setup or just prior to incubation) were held for 4, 8 or 24 hours at both room temperature and 4ºC prior to incubation. Maximal IFN-γ responses were obtained when blood was collected into lithium heparin and incubated with antigen for at least 48 hours. When the blood was stored for 24 hours prior to incubation, storage at 4ºC resulted in lower IFN-γ responses than storage at room temperature; in this case adding the antigen at the time of blood collection resulted in greater IFN- γ responses. While the findings of maximal IFN-γ response with lithium heparin and storage of blood at room temperature are consistent with previous studies in cattle, maximal IFN-γ responses in sheep were obtained by harvesting plasma at 48 hours rather than 16 to 24 hours as is recommended by the manufacturer. The results also suggest that if incubation is to be delayed, collection tubes containing antigen and media supplementation may improve IFN-γ responses.