Common measures of immune function vary with time of day and sampling protocol in five passerine species.

Ecological immunology is a rapidly growing field of study that focuses on understanding variation in immune systems across species and how this relates to species ecology and evolution. Newly developed field methods aimed at studying variation in immune function in a field setting have yielded many insights. Nonetheless, there continues to be much debate regarding the interpretation of field measures of immune function. There is substantial evidence to suggest that handling stress could introduce variation into measures of immune function, yet no study has examined the impacts of incremental changes in handling times under 30 min on immune measures. Nor has any study examined variation in immune function with time of day, though other physiological measures, including glucocorticoids known to impact immune function, vary with time of day. Here, I used observational field data to test the hypothesis that innate immune function varies with handling stress. Furthermore, I tested the hypothesis that innate immune function changes over the course of the day. I show that measures of innate immune function vary with (1) handling stress over short time periods typical of sample collection in the field, and (2) the time of day that an individual is sampled. I discuss these findings from an ecological perspective and suggest that the observed variation is not random, but is likely to have important adaptive functions. I end with a summary of the practical implications of these findings for field studies of ecological immunology.