The Shack-Hartmann wavefront sensor (SHWS) is often operated under the assumption that the sensed light can be described by a single wavefront. In biological tissues and other multi-layered samples, secondary wavefronts from axially and/or transversely displaced regions can lead to artifactual aberrations. Here, we evaluate these artifactual aberrations in a simulated ophthalmic SHWS by modeling the beacons that would be generated by a two-layer retina in human and mouse eyes. Then, we propose formulae for calculating a minimum SHWS centroid integration area to mitigate these aberrations by an order of magnitude, potentially benefiting SHWS-based metrology and adaptive optics systems such as those used for retinal imaging and microscopy.