Little is known about how design and testing methodologies affect the macroinvertebrate communities that are held captive in mesocosms. To address this knowledge gap, we conducted a 32‐d test to determine how seeded invertebrate communities changed once removed from the natural stream and introduced to the laboratory. We evaluated larvae survival and adult emergence in controls from 4 subsequent studies, as well as corresponding within‐river community changes. The experimental streams maintained about 80% of the invertebrates that originally colonized the introduced substrates. Many macroinvertebrate populations experienced changes in numbers through time, suggesting that these taxa are unlikely to maintain static populations throughout studies. For example, some taxa (Tanytarsini, Simuliidae, Cinygmula sp.) increased in number, grew (Simuliidae), and possibly recruited new individuals (Baetidae) as larvae, while several also completed other life history events (pupation and emergence) during the 30‐ to 32‐d studies. Midges and mayflies dominated emergence, further supporting the idea that conditions are conducive for many taxa to complete their life cycles while held captive in the experimental streams. However, plecopterans were sensitive to temperature changes >2 °C between river and laboratory. Thus, this experimental stream testing approach can support diverse larval macroinvertebrate communities for durations consistent with some chronic criterion development and life cycle assessments (i.e., 30 d). The changes in communities held captive in the experimental streams were mostly consistent with the parallel changes observed from in situ river samples, indicating that mesocosm results are reasonably representative of real river insect communities.