The aim of the Mont Blanc Study was to investigate the relationship between intraocular pressure (IOP), central corneal thickness (CCT), and altitude in healthy subjects. Thirty-three eyes of 33 healthy volunteers (mean age: 24.8 years, 17 females) had their IOP measured with Perkins and I-Care tonometers and their CCT using ultrasound pachymetry at three locations in Italy with different altitudes: Pavia, (PV), 77 meters above sea level (a.s.l); Courmayeur (CM), 1300 meters a.s.l; Pointe Helbronner (PH), 3466 meters a.s.l.). The measurements were performed at 9 am, 11 am, 1 pm and 3 pm (±30') in indoor settings (mean temperature of 19°C) in PV and PH. At 9 am, CCT and IOP were measured outdoor (mean temperature of -1.4°C) at PH. The mean values of the IOP curve decreased from PV to PH with the Perkins (p = 0.02) and I-Care tonometers (p = 0.001). Instead, CCT increased upon ascension from PV to PH (p = 0.01), and from CM to PH (p = 0.002). When exposed to sub-zero temperature, the IOP increased (p<0.001), while the CCT did not change (p = 0.30). The results suggest that IOP significantly decreased and CCT significantly increased upon ascension from the sea level to higher altitudes.