Eosinophilic leukocytes develop in the bone marrow and migrate from peripheral blood to tissues, where they maintain homeostasis and promote dysfunction via release of preformed immunomodulatory mediators. In this study, we explore human eosinophil heterogeneity with a specific focus on naturally occurring variations in cytokine content. We found that human eosinophil-associated cytokines varied on a continuum from minimally (coefficient of variation [CV] ≤ 50%) to moderately variable (50% < CV ≤ 90%). Within the moderately variable group, we detected immunoreactive IL-27 (953 ± 504 pg/mg lysate), a mediator not previously associated with human eosinophils. However, our major finding was the distinct and profound variability of eosinophil-associated IL-16 (CV = 103%). Interestingly, eosinophil IL-16 content correlated directly with body mass index (R2 = 0.60, ***p < 0.0001) in one donor subset. We found no direct correlation between eosinophil IL-16 content and donor age, sex, total leukocytes, lymphocytes, or eosinophils (cells per microliter), nor was there any relationship between IL-16 content and the characterized -295T/C IL-16 promoter polymorphism. Likewise, although eosinophil IL-1β, IL-1α, and IL-6 levels correlated with one another, there was no direct association between any of these cytokines and eosinophil IL-16 content. Finally, a moderate increase in total dietary fat resulted in a 2.7-fold reduction in eosinophil IL-16 content among C57BL/6-IL5tg mice. Overall, these results suggest that relationships between energy metabolism, eosinophils, and IL-16 content are not direct or straightforward. Nonetheless, given our current understanding of the connections between asthma and obesity, these findings suggest important eosinophil-focused directions for further exploration.