Off-road vehicle (ORV) traffic is one of several forms of disturbance thought to affect shorebirds at migration stopover sites. Attempts to measure disturbance effects on shorebird habitat use and behavior at stopover sites are difficult because ORV disturbance is frequently confounded with habitat and environmental factors. We used a before-after-control-impact experimental design to isolate effects of vehicle disturbance from shorebird responses to environmental and habitat factors. We manipulated disturbance levels within beach closures along South Core Banks, North Carolina, USA, and measured changes in shorebird abundance and location, as well as the activity of one focal species, the sanderling (Calidris alba), within paired control and impact plots. We applied a discrete treatment level of one flee-response-inducing event every 10 minutes on impact plots. We found that disturbance reduced total shorebird and black-bellied plover (Pluvialis squatarola) abundance and reduced relative use of microhabitat zones above the swash zone (wet sand and dry sand) by sanderlings, black-bellied plovers, willets (Tringa semipalmata), and total shorebirds. Sanderlings and total shorebirds increased use of the swash zone in response to vehicle disturbance. Disturbance reduced use of study plots by sanderlings for resting and increased sanderling activity, but we did not detect an effect of vehicle disturbance on sanderling foraging activity. We provide the first estimates of how a discrete level of disturbance affects shorebird distributions among ocean beach microhabitats. Our findings provide a standard to which managers can compare frequency and intensity of disturbance events at other shorebird stopover and roosting sites and indicate that limiting disturbance will contribute to use of a site by migratory shorebirds.