Mydriasis with muscarinic antagonists have been used routinely prior to retinal examination and sometimes prior to refractive measurements of the mouse eye. However, biometric changes during topical administration of muscarinic antagonists have not been fully investigated in mice and humans. We found that the mouse eyes treated with cyclopentolate developed a hyperopia with a reduction in both the vitreous chamber depth and axial length. In humans, prior to the cyclopentolate treatment, a 6D accommodative stimulus produced a myopic shift with a reduced anterior chamber depth, choroidal thickness and anterior lens radius of curvature and an increase in lens thickness. After the cyclopentolate treatment, human eyes developed a hyperopic shift with an increased anterior chamber depth and anterior lens radius of curvature and a reduced lens thickness. Therefore, the biometric changes associated with this hyperopic shift were mainly located in the posterior segment of the eye in mice. However, it is the anterior segment of the eye that plays a main role in the hyperopic shift in human subjects. These results further indicate that mouse eyes do not have accommodation which needs to be taken into account when they are used for the study of human refractive errors.