Many studies have demonstrated that the optic nerve sheath diameter (ONSD) is a good indicator of intracranial pressure (ICP). There are uncertainties regarding the optimal ONSD threshold, considering age and sex differences in the healthy population, and these differences could lead to uncertainties in evaluation of ONSD in pathological conditions. The aim of this prospective observational study was to investigate if age and sex could influence ONSD in a cohort of healthy Italian volunteers recruited during preanesthetic assessment for low-risk surgical procedures. The population was stratified for sex (males versus females) and for age (18–44 years, 45–64 years, and ≥65 years). The axial and longitudinal ONSD diameters were measured by two trained investigators. A significant difference in ONSD between males and females was found (median 4.2 (interquartile range 3.9–4.6) versus 4.1 (interquartile range 3.6–4.2) mm, P = 0.01), and a positive correlation between ONSD and age was found (R = 0.50, P < 0.0001). It was concluded that ONSD increases with age and is significantly larger in the healthy male population. These discrepancies should be taken into consideration when ONSD measurement is performed.