When presented the challenge of grabbing a flag on a very high flagpole (the reward is a jeep ride back to base with Carter), something no soldier has managed in over 17 years. The other soldiers scramble all over each other trying to scale the pole, but (pre-supersoldier) Rogers outsmarts his stronger squadmates by simply detaching the pole's anchor bolts and letting it fall over, rather than trying to scale it. The soldiers' and drill instructor's faces when he pulls it off are priceless.
The improvised bridge over the pit of flame has fallen in and the only way for Steve to get across is to make a superhuman leap! He backs up for all the running start he can get—and then makes a little shake of his head that clearly communicates, "Here goes nothing..."
During the procedure, Steve looks briefly taken aback when Dr. Erskine called Howard Stark, showing the latter being part of the Project Rebirth. Considering the first time Steve saw Howard's invention is when his Flying Car is Epic Fail, Steve's expression can be interpreted as This Is Gonna Suck.
Before the procedure starts, we have everyone in the lab stopping dead in their trackswhen they see Steve for the first time. Peggy's reaction is just a resigned sigh and a "let's just get this over with" expression.
However, after the procedure she's Not So Stoic. She's as gobsmacked as everyone else, and actually sneaks a feel of the dazed and newly transformed Rogers, before pulling back.
(Zola nervously studies his cell... thinking "is that blood beneath that operating table...?") Phillips: Sit down. (Phillips puts down a tray of food at a table.) Zola: What is this? Phillips: Steak. Zola: What is in it? Phillips: Cow. (Dr. Zola stares at it dubiously.) Phillips: Doctor, do you realize how hard it is to get a-hold of a prime cut like that out here? Zola: I don't eat meat. Phillips: Why not? Zola: ...It disagrees with me. Phillips: What about cyanide? Does that give you the "rummbly tummy," too?
And then Phillips promptly starts eating the steak himself.
Zola: You’re trying to intimidate me, Colonel. Phillips: I brought you dinner?
On a related note, Phillips' later description of Zola as "my new best friend" when he's debriefing the others.
Philips giving a speech to the new recruits, and as he says "We are going to win this war because we have the best men", he suddenly pauses and stumbles over the last word when he lays eyes on Rogers, a foot shorter than the other troops and still skinny and scrawny. He then ad-libs "And because they are gonna get better... much better..."
(to Steve, totally serious and terrified) "You don't have one of those, do you?"
Before jumping onto Zola's train.
Bucky: Remember the time I made you ride the Cyclone at Coney Island?
Steve: Yeah, and I threw up?
Bucky: This isn't payback is it?
Dr Abraham Erskine
Erskine proposing a toast to Rogers the night before his procedure. However, he remembers that Rogers can't have any fluids, so he snatches Rogers' glass away and drinks it down for himself.
Erskine: No, no. Wait, wait. What I am doing? No, you have procedure tomorrow. No fluids.
Steve: All right. We'll drink it after.
Erskine: No, I do not have procedure tomorrow. Drink it after? I drink it now.
And then, just before the procedure is carried out:
Steve: Did you save me any of that schnapps?
Erskine: ...Not as much as I should have.
Steve and Erskine.
"Where are you from?"
"Queens. 73rd Street and Utopia Parkway. ...before that, Germany."
"That wasn't so bad." "...That was penicillin."
The Howling Commandos
Jim Morita's indignant response (with zero accent) when Dum Dum Dugan wonders why they have to rescue a Japanese soldier as well.
"I'm from Fresno, ace!"
Becomes possible Fridge Horror if you know Fresno was at one point the site of a Japanese internment camp and the only real way to leave the camps was to join the military.
When Steve comes to rescue the prisoners from their cells, we get this priceless exchange:
Jones: Who are you supposed to be?
Steve : (awkwardly) I'm...I'm Captain America.
Falsworth: I... beg your pardon?
Keep in mind that Falsworth is British, making his confusion at the name "Captain America" even more hilarious.
Another moment from Falsworth: When Cap, Bucky and the others area bout to jump onto the train, he warns them to "mind the gap". Any Brit who's ever taken the London Underground will find this hilarious.
Jones: You sure you know what you're doing?
Captain America: Yeah. I've knocked out Hitler over two hundred times.
When Senator Brandt starts demanding answers for how a HYDRA agent got into the testing area, Colonel Phillips counters by saying "Great, why don't we start with how a German spy got a ride to my secret installation in your car."
Red Skull's first meet with Cap: "Captain America! How exciting! I'm a great fan of your films!" in an obviously-mocking tone.
Steve: I can do this all day!
Red Skull: Of course you can! Unfortunately, I am on a VERY TIGHT SCHEDULE!
When the Skull and Zola are fleeing from their exploding lab, Zola points out that the Skull's escape Triebfluegel only seats one. The Skull responds by calmly turning to Zola and handing him the keys to his car.
It's the fact that you think that the Skull is going to just leave Zola behind, but then...
Followed by the sight of Zola looking pretty gleeful as he drives away in the HydraMobile.
When Schmidt is being chewed out by the SS officers, he makes a hysterical 'One Two Three' motion with his fingers, pointing at the Nazis, just so he can adjust his death ray properly. All without pausing in his speech.
And when one of them starts to yell at him and declares he will be punished by Hitler for his treachery, Schmidt raises his eyebrows and makes a head motion to the effect of "oh really?" Then when he begins firing, one of them dodges a couple shots and he scowls, like he's thinking "Oh come on!". Finally, when he connects with the next shot, he gives a little nod, as if thinking "There that's better."
On a similar note, as Schmidt is preparing his death ray, there's a technician sitting behind the SS officers who immediately and casually walks out of the way.
Schmidt and Zola survey a destroyed HYDRA base and meet the last survivor, who says "We fought to the last man!" Zola closes his eyes and looks away before Schmidt executes the survivor. He knows what's coming.
When Steve is chasing the Hydra agent that killed Erskine. It's an awesome moment as we see first hand what the Captain is capable of. But just the entire thing is hilarious in the Agent's point of view. He remains unfazed when Cap first starts chasing him. Which is understandable since, in a normal chase a man chasing a car would eventually tire out. But when he looks again and sees that Steve is still chasing him, not only keeping up, but catching up to him, his look changes to Oh, Crap!. When he tries to escape by sub thinking he's long gone, he sees Steve catching up to him swimming, making the look seem like he's thinking "Leave me alone already!!!"