The aim of this study was to assess the effect of a single drop of hyaluronic acid on tear film thickness (TFT) in healthy subjects. Sixteen healthy subjects (eight male/eight female) aged between 20 and 36 years were included in this randomized, double-masked placebo-controlled study. One eye received a single dose of hyaluronic acid (Olixia pure(®) ; Croma Pharma, Korneuburg, Austria) eye drops, and the fellow eye received physiologic saline solution as placebo control. The study eye was chosen randomly. TFT as measured with a custom-built Fourier-domain optical coherence tomography (FD-OCT) system was the main outcome variable and measured before and every 10 min until 1 hr after topical administration. Baseline TFT was 4.8 ± 0.5 μm in the study eye and 5.0 ± 0.4 μm in the control eyes. Hyaluronic acid significantly increased TFT (p = 0.008 versus placebo) with a maximum effect 10 min after instillation (13.9 ± 11.9%). Post hoc analysis revealed that an increase in TFT was seen until 30 min after administration compared to placebo. Data in the placebo group show high reproducibility with an intraclass correlation coefficient of 0.93 and a coefficient of variation of 5.4 ± 3.3%. The data of this study indicate that hyaluronic acid increases TFT for as long as 30 min in healthy subjects. In addition, our data provide evidence that our custom-built OCT system is capable of measuring residence time of lubricants on the ocular surface.