The influences of age and gender differences on the pulse waveform in the optic nerve head (ONH) in healthy adults, using laser speckle flowgraphy (LSFG) were evaluated. We studied 908 healthy subjects (men = 701, age: 50.0 ± 9.1, women = 208, age: 49.8 ± 9.5, p = 0.76), evaluating these pulse waveform parameters: the blowout score (BOS), blowout time (BOT), acceleration time index (ATI), and the rising and falling rates. The parameters were analyzed separately for the tissue, vessels, and throughout the optic nerve head (All). All parameters were compared between genders. We investigated which independent factors for the pulse waveform in the ONH is most strongly correlated with age. All sections of the BOS, BOT, ATI, and falling rate showed a significant gender difference. A univariate regression analysis revealed that BOT-Tissue showed the strongest correlation with age (r = −0.51). The factors contributing independently to the BOT-Tissue were gender, age, heart rate, mean arterial blood pressure, pulse pressure, spherical refraction, and estimated glomerular filtration rate. Among the subjects aged >41 years, the chronological changes of BOT-Tissue in the women were significantly lower than those in the men. We concluded that the pulse waveform in the ONH has clear differences between the genders and shows chronological changes.