Yes they do. 100nl is significantly harder than 50nl, and 200nl is significantly harder than 100nl.
It's not that there are significantly less fishes, but the regs become much better as you move up. Mostly much more aggressive. So if you don't adjust properly, whatever money you take from the fishes you will donate to the better regs.
On the other hand, rake becomes relatively cheaper as you get to the $3 cap for all big pots, and that means you need less of an edge to profit.