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PURPOSE To determine the relative impact of contact lens- assisted corneal cross-linking (CACXL) and standard protocol CXL (CXL) on regional corneal stiffness using Brillouin microscopy. METHODS CXL and CACXL were performed on 30 intact fresh porcine eyes (15 per group). Depth profile of stiffness variation and averaged elastic modulus of anterior, middle, and posterior stroma were determined by Brillouin maps. Corneas were cut into strips to conduct mechanical stress-strain tests after Brillouin microscopy to evaluate stiffness difference between CXL and CACXL. Each eye served as its own control. RESULTS CXL had a greater impact on corneal stiffness, with a maximum increase of 5.74% compared to 3.99% for CACXL (P < .001). CXL increased longitudinal modulus by 7.8% in the anterior, 1.7% in the middle, and -0.7% in the posterior regions compared to CACXL, which increased longitudinal modulus by 5.5% in the anterior (P < .001), 1.2% in the middle (P = .15), and -0.4% in the posterior regions (P = .60). Mechanical stress-strain tests showed that at 10% strain averaged Young's modulus was 5 MPa for CXL and 2.97 MPa for CACXL (P < .001). CONCLUSIONS Both CACXL and standard protocol CXL induced significant corneal stiffening primarily concentrated in the anterior cornea. CACXL leads to less stiffening compared with CXL. An attenuated but continuous stiffening effect can be observed through the whole cornea for both CACXL and CXL, although CACXL has a smaller stiffness gradient. [J Refract Surg. 2019;35(11):721-728.].