I've done some research on difficulty/completion criteria of this game.
Interestingly, the level layouts actually are not based on difficulty - instead, they're randomized by RNG (RAM: 0x03C8-0x03C9). The RNG runs once per frame during the difficulty selection screen (the only place it can be manipulated, by adding frames), and a few times before a new room is generated. This is what decides the random obstacle layout/height graphics in every room, and it's not tied to any difficulty setting, loop amount, or the like.
Besides that, I went through multiple loops afterwards on skill 4 and did not find any other difference (or difficulty change) between loops. Bat/enemy speed remains the same, and the amount of rooms that has to be passed remains the same. Since the only thing that changes is randomness, and not difficulty, there's no reason to run more than one loop.
Aside from that, me and EZGames69 found a few improvements near the end of the run, adding up to 73 frames saved (most of it saved with better jumps towards Smurfette). The new movie file is
here.
EDIT: Another improvement, using better RNG manipulation for the level layouts:
13 frames saved
