ABSTRACT Clinical relevance Scleral lens (SL) compression on the conjunctiva/episclera during wear may alter adjacent structures related to aqueous humour drainage. Background The aim of this work was to assess short-term changes in the four quadrants of trabecular iris angle (TIA) and anterior chamber parameters during SL wear, using anterior segment optical coherence tomography (AS-OCT). Methods Prospective study was conducted involving 16 healthy subjects with a mean age of 28 ± 5 years. Two SL of diameter 15.80 mm (L1) and 16.80 mm (L2) with the same parameters were used for 2 hours in different days. The central fluid reservoir (FR), the TIA in superior, inferior, nasal, temporal quadrants, and anterior chamber parameters; anterior chamber depth (ACD), anterior chamber width (ACW), anterior chamber area (ACAr) and anterior chamber volume (ACV) were measured with AS-OCT. These measurements were performed immediately after SL application (0 h), one hour (1 h) and two hours (2 h) of SL wear. Results TIA decreases significantly in the superior quadrant at 2 hours of L2 wear (−5.19 ± 7.79º) (p = 0.04). In this sector, differences were also observed between the changes induced by both lenses during the 2 hours of wear (p = 0.04) and between first and second hours of wear (p = 0.04). ACD was significantly lower with L1 (−0.09 ± 0.14 mm) relative to L2 (0.00 ± 0.06 mm) between immediately after the application and 2 hours of wear (p = 0.02). A significant reduction in ACAr at 1 hour (−0.65 ± 0.75 mm2) (p = 0.01) and 2 hours of wear (−0.81 ± 1.15 mm2) (p = 0.04), as well as in ACW at 2 hours (−0.11 ± 0.14 mm) (p = 0.02) compared to immediately after application of L1 was found. Conclusion Small changes in TIA and anterior chamber occur during two hours of SL wear, but these changes are of limited clinical relevance in healthy subjects.