OBJECTIVE To evaluate changes in subfoveal choroidal thickness (SFCT) and peripapillary choroidal thickness (PPCT) after acute intraocular pressure (IOP) elevation provoked by a dark room prone provocative test (DRPPT). METHODS The prospective cohort study included 114 eyes from 65 individuals who had an IOP elevation ≥2 mm Hg during the DRPPT. The participants stayed in a dark room for 2 h with the forehead placed on a desk. At baseline and within 5 min after the end of the DRPPT, tonometry and enhanced depth imaging by spectral-domain optical coherence tomography were carried out. RESULTS During the DRPPT, IOP increased by 10.1±10.9 mm Hg, SFCT decreased significantly (p<0.001) from 280±80 µm to 267±76 µm and PPCT decreased significantly (p<0.001) from 177±74 to 169±70 µm. In multivariate analysis, a more marked SFCT thinning was associated with higher IOP increase (p<0.001) and shallower anterior chamber depth at baseline (p=0.01). In a similar manner, a higher PPCT change was correlated with a higher IOP increase (p<0.001), and a thicker choroidal thickness at baseline (p<0.001). CONCLUSIONS Choroidal thickness in the subfoveal region and in the peripapillary region decreased parallel to an acute increase in IOP in individuals 2 h after a dark room test. Choroidal thickness depends on the actual IOP, which may be noted when choroidal thickness is measured.