well, of course PHP reads 'all' the contents of the file you specify in require or include before moving ahead.
it would really depend on your server spec. if it only has about 32MB ram and 200 Mhz, and require file is about 2000 lines of code.. then it might show some performance loss 🙂
but normally, the difference is not so noticeable at all... may be few milliseconds or so..