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Measured corneal biomechanical properties are driven by intraocular pressure, tissue thickness, and inherent material properties. We demonstrate tissue thickness as an important factor in the measurement of corneal biomechanics that can confound short-term effects due to UV riboflavin cross-linking (CXL) treatment.We isolate the effects of tissue thickness on the measured corneal biomechanical properties using optical coherence elastography by experimentally altering the tissue hydration state and stiffness.Dynamic optical coherence elastography was performed using phase-sensitive optical coherence tomography imaging to quantify the tissue deformation dynamics resulting from a spatially discrete, low-force air pulse (150-μm spot size; 0.8-millisecond duration; <10 Pa [<0.08 mmHg]). The time-dependent surface deformation is characterized by a viscoelastic tissue recovery response, quantified by an exponential decay constant—relaxation rate. Ex vivo rabbit globes (n = 10) with fixed intraocular pressure (15 mmHg) were topically instilled every 5 minutes with 0.9% saline for 60 minutes and 20% dextran for another 60 minutes. Measurements were made after every 20 minutes to determine the central corneal thickness (CCT) and the relaxation rates. Cross-linking treatment was performed on another 13 eyes, applying isotonic riboflavin (n = 6) and hypertonic riboflavin (n = 7) every 5 minutes for 30 minutes, followed by UV irradiation (365 nm, 3 mW/cm2) for 30 minutes while instilling riboflavin. Central corneal thickness and relaxation rates were obtained before and after CXL treatment.Corneal thickness was positively correlated (R2 = 0.9) with relaxation rates. In the CXL-treated eyes, isotonic riboflavin did not affect CCT and showed a significant increase in relaxation rates (+10%; P = .01) from 2.29 ms−1 to 2.53 ms−1. Hypertonic riboflavin showed a significant CCT decrease (−31%; P = .01) from 618 μm to 429 μm but showed little change in relaxation rates after CXL treatment.Corneal thickness and stiffness are correlated positively. A higher relaxation rate implied stiffer material properties after isotonic CXL treatment. Hypertonic CXL treatment results in a stiffness decrease that offsets the stiffness increase with CXL treatment.