It's probably no secret by now that you can have more than 80. But what's the actual limit? I decided to do a bit of investigating.
Firstly, Lemmix will recognize ANY value (as long as it fits in the two bytes the level format allows for). You can have a level with 65535 lemmings if you want.
But how well does this work in practice? Well. At around 150 lemmings, Lemmix starts to lag when in fast forward mode. Around 400, it starts to lag even at normal speed - though it's not jerky at all, it just runs at a reduced framerate (the timer does indeed slow down proportionally, so it won't cause you to run out of time prematurely). I suspect this lag actually gradually increases with more lemmings rather than having a defined starting point, but for me, exactly 400 lemmings is when it first becomes noticable.
So if we're willing to put up with it running slow (or we have a supercomputer in our bedroom), what can Lemmix actually handle? Well, first thing to consider is the interaction with the time limit - a 9 minute level with release rate 99, the time would run out exactly as the 2283rd lemming comes out. But of course - unlike the DOS game, Lemmix can handle time limits of up to 99 minutes with no problem, and up to 255 minutes with only a minor graphical glitch (specifically, it'll only show the last two digits of the remaining minutes). It should be noted that by this point, Lemmix is running at about 4 FPS, and the only practical way to advance time is using the time skip by pushing spacebar.
After around half an hour (by the game clock), we're at about 8200 lemmings, and if we unpause the game, it runs at what appears to be less than a single frame per second. But we've already said that the practical limit lies at around 400 lemmings, beyond that it's just a matter of "screw practical, this is now just about what's physically possible".
After an hour by the game clock, we're just a few lemmings shy of 15300. At this point, even with the game paused and only using the spacebar's 10 second skip, lag is noticable - actually, this was true from about 10,000 lemmings or so. I guess that's to be expected when processing and drawing that many lemmings.
At about 65 minutes 20 seconds, we pass the 16384 lemmings mark. (Some of you may know why that number is significant.) Nothing notable happens - but there again, if anything's going to happen, the most likely point is 32768 (or even more so 65536, but that's one lemming more than the LVL format allows you to set).
After two hours by the game's clock, we're at about 30300 lemmings. About eight and a half minutes after that, we hit and pass 32768 without problems. Surprisingly, even at this point, the Lemmix player is only using about 13MB of memory according to Task Manager.
I think, short of running into memory issues (which is unlikely, given the low amount being used), this should go all the way to 65535 without problems (other than lag). I see no reason why any value inbetween is likely to cause problems.
So, are there any issues with having >80 lemmings, other than lag? The short answer is, there can be.
The long answer is, up to 100, there is no issues. Beyond this, think about the way the game gives you results - as a percentage. Now obviously, once you go past 100, each lemming is worth less than 1%, but the game only displays whole numbers. It WILL calculate correctly your percent and round it down as normal, but here's something important to consider - Lemmix does not compare the number of lemmings you've saved to the level's requirement, it compares the percentages. Say for example, if you create a level with 200 lemmings and set the requirement to 199, this will display as 99%. However, if you only save 198 lemmings, this will also display as 99% - and will indeed register as passing the level. Obviously, we can't say whether DOS works the same way (other than by reverse engineering it and looking at the actual mechanics, as opposed to experimentally) due to the 80 lemming limitation - possibly if some other platform that uses percentage can handle more than 100 lemmings (perhaps Amiga? None of the levels natively have >100, but what happens if you edit a level to have more?), you could try it...
The other consideration is of course, what a level editor will allow you to set. I haven't tried seeing what Lemmix or jLevelBuilder will allow, but LemEdit won't let you set more than 80 lemmings. Of course, this can be averted by using a hex editor, and perhaps if needed, Mindless's tools to compress them into a DAT file instead of using the editor's built in feature.
(Don't freak out guys, I'm not going to slip a 65535-lemming level into LP5. =P I will be using 100 rather than 80 as the general upper limit, and may exceed it for a couple of levels - but even those would be WELL under the 400 mark, probably around 200 or so at the most.)