To compare the biomechanical properties of the cornea after epithelial removal in eyes with keratoconus undergoing corneal cross-linking. Prospective interventional case series at a university hospital tertiary referral center. Corneal biomechanical properties were measured in patients with keratoconus undergoing corneal cross-linking, immediately before and after epithelial debridement by using a dynamic ultrahigh-speed Scheimpflug camera equipped with a noncontact tonometer. The study comprised 45 eyes of 45 patients with a mean age of 19.6 ± 4.9 years (range 14-34). The cornea was found to be 23.7 ± 15.7 μm thinner after epithelial removal (P < 0.01). Corneal stiffness was reduced after epithelial removal as demonstrated by a significant decrease of parameters such as stiffness parameter A1 (12.31, P < 0.01), stiffness parameter-highest concavity (2.25, P < 0.01), A1 length (0.13 mm, P = 0.04), highest concavity radius of curvature (0.26 mm, P = 0.01), highest concavity time (0.22 ms, P = 0.04) and an increase in A1 velocity (-0.01 m/s, P = 0.01), A1 deformation amplitude (-0.03 mm, P ≤ 0.01), A1 deflection length (-0.32 mm, P < 0.01), A2 deformation amplitude (-0.03 mm, P = 0.01), and A2 deflection length (-1.00 mm, P < 0.01). There were no significant differences in biomechanical intraocular pressure (0.15 mm Hg, P = 0.78), deformation amplitude (0.03, P = 0.54), maximum inverse radius (-0.01 mm, P = 0.57), and whole eye movement length (-0.02 mm, P = 0.12). Dynamic ultrahigh-speed Scheimpflug camera equipped with a noncontact tonometer offers an alternative method for in vivo measurements of the epithelial layer's contribution to corneal biomechanical properties. Our results suggest that corneal epithelium may play a more significant role in corneal biomechanical properties in patients with keratoconus than previously described.