For the same diamond blade extension, uphill (centripetal) radial keratotomy incision direction achieves greater depth and consequently greater refractive effect than downhill (centrifugal) incisions. To determine which factors may account for this difference, two uphill and two downhill incisions were made with a double-edged diamond blade set to 90% central pachometry in 26 human donor eyes at 15 or 60 mm Hg. Uphill incisions made with the perpendicular blade had greater mean incision depth than downhill incisions made with the oblique blade at 15 mm Hg (83.6 ± 3.9% and 68.2 ± 5.2%) (p<0.0005) and at 60 mm Hg (86.3 ± 3.1% and 79.7 ± 1.7%) (p<0.0005). Uphill and downhill incisions both made with the perpendicular blade had equal depth (85.4 ± 4.9% and 83.7 ± 3.5%) (p>0.1). The perpendicular blade edge created a straight, and the oblique edge an S- or J-shaped, histological incision configuration. Corneal profile pictures taken during each incision showed the knife to tilt opposite of the incision direction and to move at a constant angle to the limbal plane, producing a smaller optical clear zone (OCZ) in the posterior stroma than intended with uphill incisions. Greater refractive effect with uphill incisions may be explained by the perpendicular blade being more effective in incising corneal lamellae, and the creation of a smaller posterior OCZ. Intraocular pressure variations during surgery may affect achieved incision depth of downhill, but not of uphill, incisions.