Evaluation of ex vivo innate immune responses requires the transport of blood to a laboratory for analyses. Storage temperature and duration are highly variable in the published literature. The objective of the present study was to establish recommended storage temperature and duration for blood before analysis of several ex vivo innate immune responses in vitro. Peripheral blood was collected from 8 clinically healthy, 12-mo-old Holstein heifers and stored either on ice or at ambient temperature for 1.5, 3.5, 5.5, 7.5, 9.5, and 24. h before conducting analyses for lipopolysaccharide stimulated whole blood tumor necrosis factor-α (TNF-α) secretion, neutrophil L-selectin expression, and neutrophil phagocytic and oxidative burst capacities when exposed to an Escherichia coli. L-Selectin expression was greater in neutrophils stored on ice as early as 1.5. h after blood collection. In addition, when neutrophils were stored on ice, L-selectin expression at 24. h was not different from that at 1.5. h after blood collection. Measures of innate immune cell function, including whole blood TNF-α secretion and neutrophil phagocytic and oxidative burst abilities, were decreased in blood stored on ice. Both TNF-α secretion and neutrophil oxidative burst intensity did not differ at 24. h compared with 1.5. h after blood collection when blood was stored at ambient temperatures. These data indicate that both storage temperature and duration influence measures of innate immune responses. Therefore, storage durations should be minimized and storage protocols kept consistent to reduce the influences of storage conditions on experimental results.