To study central corneal pachymetric variations during corneal collagen cross-linking (CXL) treatment with the use of riboflavin and ultraviolet A irradiation (UVA). Prospective, noncomparative, interventional clinical study. Fifteen keratoconic patients (19 eyes) were enrolled. All patients underwent riboflavin-UVA-induced corneal CXL. Intraoperative central corneal thickness (CCT) measurements using ultrasound pachymetry were performed during the procedure. Measurements were obtained after epithelial removal, after riboflavin drop instillation, and every 5 minutes (6 interval times) during UVA irradiation (30 minutes). Central corneal thickness measurements. Mean patient age was 26.9+/-6.5 years (range, 17-40 years). Ten were male and 5 were female. Mean preoperative CCT was 458.5+/-21.5 microm (range, 427-494 microm; 95% confidence interval CI, 448-467 microm) and 415.7+/-20.6 microm (range, 400-468 microm; 95% CI, 406-426 microm) before and after epithelial removal, respectively. There was a statistically significant decrease (mean, 75 microm) of CCT between the epithelial removal interval (415.7+/-20.6 microm; range, 400-468 microm) and at the end of riboflavin solution instillation (340.7+/-22.9 microm; range, 292-386 microm; P<0.001). There was no statistically significant change in CCT during irradiation (P>0.05). There was no statistically significant difference between preoperative and 1-month postoperative endothelial cell count (preoperative, 2780+/-197 to 1-month postoperative, 2713+/-116; P = 0.14). No intraoperative, early postoperative, or late postoperative complications were observed in this patient series. During corneal CXL with the use of riboflavin and UVA irradiation, a statistically significant decrease of CCT was demonstrated.