Recent versions of PHP have a cache of filenames for knowing the real path of files, and require_once() and include_once() can take advantage of it
require_once()
include_once()
You've probably already found this, but for those who come across this question, you can use realpath_cache_size() and realpath_cache_get() to figure out how much of the realpath cache is being used on your site and tune the settings accordingly.