Grif of Red vs. Blue spends the last moments of the third season screaming "No-!" (seemingly for hours, as said by Donut.), after the Red Team returns to Blood Gulch after an endevour with O'Mally. This extends to the the entire first episode of the fourth season, with lots of ridiculous plans (devised by Sarge, who else?) that attempt to force him off the cliff he's perched on. The plans are, in order, allowing him to scream until he passes out.
Grif: NOOOOO! Sarge: Simmons! Find a way to get Grif back down here right now! Simmons: Alright. HEY GRIF! SHUT THE FUCK UP AND GET DOWN HERE! WE NEED TO CHECK OUT THE BASE! Grif: NOOO! NOOOOOO! NOOOOOOOOOOO! Simmons: I don't think he's listening, sir. Grif: NOOO! THAT TIME I WAS ANSWERING YOUR QUESTION! NOOOOO! Sarge: That does it, Simmons, go get him down now. Donut: Sarge, if he keeps screaming like that, he'll pass out, and fall off the cliff! Sarge: Cancel that order Simmons! Grif: NOOOOOOOO! Sarge:(sighing) Donut, get me a sniper rifle.
Gets played much more seriously later in the series: in Season 8's penultimate episode, from Church when The Meta stabs Tex and extracts her into an AI memory unit; in Season 10's episode 16, from Alpha-Church, when Sigma (as The Director) tells him Tex is dead; and episode 19 of Season 10, from Carolina as the newly-"born" Meta rips the AIs Eta & Iota out of her skull, and from Tex when The Meta proceeds to throw Carolina over a cliff to her (presumed) death.
Strong Mad gets one of these in an encounter with Senor Cardgage on Homestar Runner.
Marzipan gets to say one after being forced to become Coach Z's girlfriend in "Doomy Tales of the Macabre". Since she read the cue card incorrectly, her first attempt was pronounced with a short "o" and sounds more like "moo" with an "n" than "No!"
In 'The Camping Webisode' of DSBT InsaniT, Koden does this when he discovered the groups campsite has been ruined, and Duck when Martha accidentally pops Balloon.
In "Gelatin Swingsets", the Cutie Mark Crusaders are dismayed to discover their coupon for a free gelatin swingset has been taken away, and Scootaloo lets loose a Big No. But since she only speaksscat, it comes out as:
In the opening of "The Penny and Clyde Show", the big no pulls double duty as an expression of dismay and and answer.
Pinkie: Nooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo... Applejack: Dammit, Pinkie, would you just shut up? Pinkie: NooooooooooooooooooooooooooooooooooooooooooooooŚ
Sage from "Shadows Of The Past" in episode 20 does this after Eieon mentions they should get Doc and Pinky to help them study the giant computer they found in the humming box. Justified after the ordeal they just went through with two assassins and a giant killer mech one year old freak child. It Makes Sense in Context