Measuring optical density with/without re-suspending cells
Definitely (2). Clumps of cells will cause a lot of noise in your absorbance measurements (depending on whether the light beam hits a clump or some empty space).

I don't know what your microbe is or what growth medium you're assaying on, but typically it's very important to maintain the culture well-mixed at all times, not just during measurement. If your cells clump to the bottom, you will not get the same aeration as you will if they're well-mixed, and accessibility of other nutrients could potentially be limited as well. Generally the most controlled condition is to maintain good mixing at all times, but certainly this is organism- and medium-dependent.