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Aquatic invertebrates are excellent indicators of ecosystem quality; however, choosing a sampling method can be difficult. Each method and associated protocol has advantages and disadvantages, and finding the approach that minimizes biases yet fulfills management objectives is crucial. To test the effects of both sampling methods and sample handling-i.e., to composite samples or leave them as replicates-we collected aquatic invertebrates from the Niobrara River at Agate Fossil Beds National Monument, Nebraska, using three methods and two sample handling protocols. We compared aquatic invertebrate assemblages collected with a Hester-Dendy multi-plate sampler, Hess sampler, and a D-frame dipnet. We calculated six common bioassessment metrics from composite (combined) and replicate (separate) samples. Hess samples contained the highest taxonomic richness (capturing 77% of all taxa observed) and dipnet samples the least (47%). Hester-Dendy samples had the greatest proportion of Ephemeroptera, and Ephemeroptera, Plecoptera, and Trichoptera (EPT). Dipnet samples had the lowest evenness values. In terms of sample handling, composite samples had inflated richness, diversity, and evenness compared with replicate samples, but bioassessment metrics calculated from proportions or averages (i.e., Hilsenhoff's Biotic Index and the proportion of EPT taxa) did not differ between them. The proportion of invertebrate groups from composite samples were not statistically different among sampling methods, but several groups differed between replicate samples collected by different methods. Ultimately, we recommend collecting replicate samples with a Hess sampler when the goal of the study is to detect ecosystem change, among locations or differences in variables of interest.