Outdoor education (OE) stream classes provide students with an opportunity to gain hands-on experience with sampling methods for evaluating stream water quality. Trampling by students as a result of stream classes may disrupt the substrate and negatively impact aquatic macroinvertebrates. The impact of student-induced trampling in headwaters as a result of stream classes on aquatic macroinvertebrates has not been evaluated. Our aim was to document the short-term macroinvertebrate responses to an experimental disturbance that simulated the impacts of trampling by students in riffles within small headwater streams. We measured hydrologic variables, visually estimated substrate composition and sampled aquatic macroinvertebrates within control and experimental riffles in three agricultural headwater streams in central Ohio one day prior to experimental disturbance, immediately after disturbance and one day after disturbance. Hydrologic variables and substrate type did not differ daily or between riffle types. Macroinvertebrate abundance, percentage of Ephemeroptera Plecoptera Trichoptera and percentage of Leuctridae increased after experimental disturbance, while diversity, evenness, percentage of clingers and non-metric multidimensional scaling (NMS) axis 1 site scores declined after disturbance. Macroinvertebrate diversity, percent clingers and NMS axis 1 site scores were lower in experimental riffles than control riffles. None of the macroinvertebrate response variables exhibited a significant interaction effect of day × riffle type that is indicative of an effect of the experimental disturbance. Our results suggest the one-time use of an undisturbed riffle within an agricultural headwater stream for an OE stream class is not likely to impact aquatic macroinvertebrates.