The purpose of this study is to quantify the impact of enzyme activity on the vitreous humor structure over time to understand the mechanical characteristics of the vitreous humor gel. Changes in the mechanical behavior of the vitreous occur due to many reasons including aging, which may lead to many vitreoretinal diseases. The degeneration process of the vitreous has been studied; however, in situ experimental procedures to validate the existing hypotheses are limited. We examined thirty-eight porcine eyes using in situ rheological creep tests to measure the mechanical properties of the vitreous humor of the eyes prior to, 1 h and 24 h after the intravitreal injection. Eyes in one group were injected with collagenase type II solution and eyes in the control group were injected with phosphate buffered saline solution (PBS) with calcium and magnesium chloride. Prior to the injection, viscosity and creep compliance intercept values between both groups were not statistically different. At 1 h and 24 h after the injection, vitreous properties in the eyes from the first group showed a statistically significant increase in the J intercept values (representing the inverse of elasticity) compared to the control group. In addition, 1 h and 24 h after the injection, vitreous viscosity was lower in the eyes from the first group than in the eyes from the control group. These findings are a foundation for future studies on the effectiveness of intravitreal drugs that modify the mechanical properties of the vitreous humor.