Yes, it definitely makes a difference. On my vbulletin forum, I have it set for 20 minutes. If I change the time to 40 minutes it makes it look like there are double the amount of people on. Unless you can see the who's online section, you can't tell what each guest is doing and when their last activity is.
At any given time, I have 50 Yahoo spiders on my forum. They don't always count in the forums themselves because they search profiles, images, and really weird URLs (part of that has to do with Zoints I have installed).