Revealing mistake: As the guy is being pinned down by the giant hooks you can see for over a minute he can simply escape and slip his hands out. (00:52:30 - 00:53:30)
Factual error: In his crude attempt to replicate the Human Centipede surgical procedure, Martin employs several handyman tools, including a carpenter's staple gun (using T50 staples as sutures). Problem is, carpenter's staples would never join soft human body tissues, they'd pull right out with little effort (T50 staples only join to very solid base materials, such as wood). Martin's victims, hysterical as they were, could have freed themselves from the T50 staples almost immediately.
Continuity mistake: At the end of the movie, the bloody handprint on the car changes entirely the second time it is shown. (02:02:45)
Other mistake: In the hospital, Barrett had his right arm in a sling and his lower left leg in a cast. When the police/ detectives were trying to find Barrett, one detective said Barrett could not have driven himself somewhere because he had "a cast on his leg." The cast was on the left leg, so Barrett could have driven using his right leg, as usual. Maybe he couldn't have driven with a cast on his right arm, but the detective gave the wrong location of the leg cast. (00:50:30 - 01:13:45)
Factual error: The young priest sees images of a red-eyed horse, but later, it is referred to as a mule, which looks much more like a donkey.
Visible crew/equipment: As Max opens the bathroom door on the plane, a reflection of the crew is visible in the door. (00:50:10)