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Stable isotope analyses offer a useful means for quantifying ecological niche dimensions, though few studies have examined isotopic response of an ecological community with respect to resource gradients such as fluctuations in prey availability. Stable carbon and nitrogen isotopes were measured for Lake Michigan salmonines and their prey collected from 2014 to 2016. Bayesian ellipse and mixing model analyses were used to quantify isotopic niche characteristics and diets, respectively, among species and years. During the three-year study period, abundance and size structure of preferred alewife prey changed substantially and offered an opportunity to explore predator isotopic niche response and diet shifts along a prey resource gradient. Results suggested increased reliance on alewives, especially small alewives, over the study period and were consistent with greater availability of this prey. However, differential use of alewife size classes and alternative prey sources by salmonine predators was apparent, which suggested possible resource partitioning. Characterization of ecological niche overlap using stable isotopes likely requires consideration of shared resource availability as well as specific prey and habitat preferences.