[geofft]

What's the actual effect of this? Negative filesystem lookups are cached, and I believe they're cached forever on local filesystems (since you know if anyone's writing to the local filesystem), so the overhead is just that of a couple of syscalls. That feels like it's small enough that you really want to benchmark to see if it matters for you.

[combatentropy]

No effect, on my server, a $5-per-month Digital Ocean droplet. Using the program called Apache Bench (ab) to retrieve a file four directories deep: /a/b/c/d/e.html

With .htaccess files turned off (AllowOverride None), I got 282 requests per second. With .htacess files on (AllowOverride All) I got 286 requests per second (Yes, actually a few more hits per second with .htaccess turned on, but the results swing from test to test by a few percent anyway).