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To assess whether fluorescein dye in the tear film results in a clinically relevant change in central corneal thickness (CCT), as measured with Scheimpflug imaging (Pentacam HR; Oculus, Wetzlar, Germany). The study was conducted at Moorfields Eye Hospital NHS Foundation Trust.In this prospective, randomized, controlled, single-center study on healthy volunteers, CCT was measured using rotating Scheimpflug imaging and partial coherence interferometry technique. After 3 baseline measurements of both eyes, a drop of 0.25% fluorescein with 0.5% proxymetacaine hydrochloride dye, as used for measuring intraocular pressure with applanation Goldmann tonometry, was instilled in the first eye and measurements were repeated after 1 minute and 5, 10, 20, 40, and 60 minutes. A fluorescein drop was then instilled in the second eye, and 1 measurement was performed after 1 minute. Immediately afterwards, the second eye was rinsed with 2 ml of balanced saline solution and the CCT measurement was repeated. On a different day, a Schirmer II test was performed.Thirty eyes of 15 volunteers were included in this study. The average CCT measured with Scheimpflug imaging was 539.1 μm (SD, 32.2 μm), and after instilling fluorescein, CCT values increased by 46.6 μm (SD, 11.4 μm; P < 0.01, paired t test). In 10 of 15 subjects, the fluorescein layer was still present 40 minutes after instilling the drop. Rinsing the eye decreased the fluorescein layer by 49.6% and a value of 24.8 μm (SD, 16.8 μm).Because CCT measurements are critical for corneal refractive surgery, Scheimpflug imaging should not be performed after use of fluorescein dye for staining of the corneal epithelium or for applanation tonometry.