The heterophil to lymphocyte (HL) ratio in the peripheral blood is increasingly being recognized as a reliable indicator of stress in birds. In this study we examined whether HL ratio, as well as the proportion of heterophils and lymphocytes, varied systematically with sampling time, date, age, climate conditions or with measures of maternal investment in female tree swallows (Tachycineta bicolor) over three breeding seasons. We found that HL ratios showed significant annual variation, which appeared to be driven by annual changes in the proportion of heterophils. HL ratios were higher among those females laying larger clutches, suggesting a potential cost of reproduction. Variation in body condition also appeared to affect stress levels of females, as decreases in body condition were associated with elevated HL ratios. Among females that we sampled over multiple breeding seasons, we were unable to detect significant repeatability for both HL ratios and proportion of heterophils, although proportion of lymphocytes showed low but significant repeatability within individuals. We therefore suggest that caution should be exercised in using these measures for illustrating the inherent quality or health of individuals over time frames beyond the current breeding attempt, or as predictors of future reproductive potential.