Subject Sixteen's Sanity Slippage commentary at a few points in the Glyph puzzles. It ends with him declaring that he's unable to deal with all the pain he's lived through via the Animus - "The guillotine cuts my neck, the bullet hits my chest, the water fills my lungs..."
Also his comment of "the woman cries as I fuck her". It's small, but it's that moment it hits that, instead of the noble Assassins like Altair, Ezio and Connor, Desmond could have relived a monster's memory...
A couple of the videos Shaun makes for Ezio's targets:
Francesco de'Pazzi's ends with a shot of Giovanni Auditore after he's been hanged.
Vieri's isn't much better, as it ends with him serving a rival and his family a meal "to die for."
Silvio Barbarigo's mentions how his uncle apparently died in bed - and we see an image of two courtesans approaching him with knives, while Silvio watches from the doorway.
A few of the Glyph puzzles, if you think about it enough. The Templars have a longer reach than you know, and fingers in every pie.
"Hat Trick" - another "find the Apple" puzzle, except the "you're getting closer" is the sound of a heartbeat picking up speed. When you find it, it changes to a "patient is flatlining" beep.
"THEY BURNED JOAN ALIVE" - cue painting of Joan being burned at the stake, accompanied by horrifying scream.
Topsy, especially since this was something Edison actually did to ruin Tesla.
Minerva's warning about the First Disaster - especially after you've played Revelations.
The Poison Blade. It's victims begin feeling uneasy at first, but before long they start trashing on the ground and begging for help. No wonder Alta´r called poison a coward's tool!
Savonarola's execution in the Bonfire of the Vanities DLC. Sure, he kind of had it coming, considering what an asshole he was, but it is still really painful to watch and listen to. He is bound to a stake and a fire is started directly underneath his feet. Even Ezio thinks it is to gruesome a death for anyone to suffer and puts him out of his misery with a quick, albeit brutal, neck-stab. And for extra discomfort, if you fail to do this, you get to listen to Savonarola's frightened prayer turning into agonized screaming as he is cooked. Nice job there.
In the final Assassin's Tomb, you have to free run to hit a series of switches across a large underground lagoon...a lagoon that happens to house a very large and irritable giant squid that takes a swing at Ezio in a cutscene. Sure, nothing actually happens if you fall in the water, but just try and stop yourself from cringing when you do.
One of the easter eggs involves looking into murky water for at least forty seconds. Upon doing this, a massive octopus-like creature swims by, glowing orange eyes fixing on you for a second. Try it again, and it attacks you with a tentacle, just barely missing its mark.
During one of the glyph puzzles ( Martyrs to be accurate ), the player drags a fire-like icon over an image. They are then (likely) caught off-guard when the ambient silence maintained through most of the puzzle sections is abruptly interrupted by the sound of agonized screaming, with an image of Joan of Arc burning at the stake, with the words " THEY BURNED JOAN ALIVE . THEY TOOK IT." over it. This disappears quickly to move onto the next part of the puzzle, but when you're playing all on your own in a dark basement with the volume up, it really screws with your head... Then again, the stock scream sound might make other players (probably those a little less aurally immersed) consider this Narm instead.
The one when you hear Subject 16 talking about killing and fucking people and completely losing his mind.
The Martyrs and Hat-trick puzzles are noteworthy, because when you're going through the puzzle, the sound it makes isn't a beep; it's a heart beating faster and faster as you get closer to the Piece of Eden, and when you ID it - flatline.
That poor fucking elephant. It's not fake. Thomas Edison really did that. Just to smear Nikola Tesla. IRL, there's no proof that he was a Templar, but there's the proof he was a dick.
The "acquittal" of Giovanni, Federico and Petruccio Auditore... because it's one thing to see already-dead hanging corpses in a video game, it's a completely whole 'nother to witness the execution without a discretion shot, except for Petruccio, and if you watch Francesco de' Pazzi's video, it ends with a close-up of the slain Giovanni's face, the noose still around his neck.
Ezio slowly pushing his hidden blade into Checco Orsi's throat. It makes what Alta´r did to Majd Addin look friendly by comparison.
The Doctor for every courtesan in Roma, especially Fiora Cavazza who, despite all the killings she participated in and madmen she encountered at this moment, is scared out of her mind when she has to meet him for Cesare. Thankfully for her, she realizes one second before it's too late than he's a Dirty Coward.