To investigate the diurnal variation of the pulse waveform parameters determined by laser speckle flowgraphy (LSFG) on the optic nerve head (ONH) in healthy subjects.This prospective cross sectional study was conducted at Nagoya University Hospital. We studied 13 healthy volunteers whose mean age was 33.5 ± 7.6 years. Eight pulse waveform parameters on the ONH were determined by LSFG (LSFG-NAVI) every 3 hours from 6 am to 12 pm. The intraocular pressure (IOP), systolic (SBP) and diastolic (DBP) blood pressure, and heart rate (HR) in the brachial artery were also recorded. We evaluated the diurnal variations of the parameters and compared the pulse waveform parameters to the other parameters using a linear mixed model.Of the 8 parameters, skew (P < .001), blow out score (BOS, P < .001), blow out time (BOT, P = .028), rising rate (P < .001), falling rate (P < .001), resistivity index (P < .001) had a significant diurnal fluctuation. In addition, IOP (P < .001), SBP (P = .005), DBP (P = .001), and HR (P < .001) had significant diurnal fluctuations. The BOS and resistivity index were significantly correlated with the HR (P = .009, P = .012, respectively), and the BOT were significantly correlated with the DBP and mean ocular perfusion pressure (P = .042, P = .041, respectively).We found that there was significant diurnal variation in 6 waveform parameters on the ONH in LSFG. We believe that our results highlighting diurnal variations in these waveform parameters need to be considered when interpreting pulse waveform parameter data and in understanding the precise underlying mechanism of ocular diseases such as diabetic retinopathy, retinal vein occlusion, and glaucoma.