PURPOSE. Mice are commonly used to study conventional outflow physiology. This study examined how physical factors (hydration, temperature, and anterior chamber [AC] deepening) influence ocular perfusion measurements in mice. METHODS. Outflow facility (C) and pressure-independent outflow (Fu) were assessed by multilevel constant pressure perfusion of enucleated eyes from C57BL/6 mice. To examine the effect of hydration, seven eyes were perfused at room temperature, either immersed to the limbus in saline and covered with wet tissue paper or exposed to room air. Temperature effects were examined in 12 eyes immersed in saline at 20°C or 35°C. Anterior chamber deepening was examined in 10 eyes with the cannula tip placed in the anterior versus posterior chamber (PC). Posterior bowing of the iris (AC deepening) was visualized by three- dimensional histology in perfusion-fixed C57BL/6 eyes and by spectral-domain optical coherence tomography in living CD1 mice. RESULTS. Exposure to room air did not significantly affect C, but led to a nonzero Fu that was significantly reduced upon immersion in saline. Increasing temperature from 208C to 358C increased C by 2.5-fold, more than could be explained by viscosity changes alone (1.4-fold). Perfusion via the AC, but not the PC, led to posterior iris bowing and increased outflow. CONCLUSIONS. Insufficient hydration contributes to the appearance of pressure-independent outflow in enucleated mouse eyes. Despite the large lens, AC deepening may artifactually increase outflow in mice. Temperature-dependent metabolic processes appear to influence conventional outflow regulation. Physical factors should be carefully controlled in any outflow studies involving mice.