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To evaluate changes in choroidal thickness before and after steep Trendelenburg position (STP, 40° head-down) using automated segmentation software to analyse swept-source optical coherence tomography (SS-OCT) data.The eyes of 20 healthy volunteers underwent a three-dimensional wide scanning protocol with SS-OCT (Topcon, Tokyo, Japan) at baseline, immediately after STP was initiated, and 5, 10 and 15 min after STP; blood pressure and heart rate were measured concurrently. The predictive mean difference was calculated using a generalised linear mixed model that adjusted for potential confounders.Mean choroidal thickness significantly and transiently increased immediately (268.18±9.24 μm, p<0.01) and 5 min (264.25±9.30 μm, p=0.03) after STP, relative to baseline (256.51±9.20 μm). However, choroidal thickness decreased by 10 min (262.51±9.34 μm, p=0.15) and 15 min (261.38±9.40 μm, p=0.37) after STP. Mean arterial pressure also transiently increased from baseline (78.2±1.2 mm Hg) immediately after STP (79.9±1.1 mm Hg, p=0.01), but normalised by 5 min after STP (p>0.05 for all comparisons ≥5 min).Choroidal thickness significantly but transiently increases after adopting STP, as evaluated by automated layer segmentation analysis of SS-OCT data.