well, for anyone interested.. i ran some tests..
i had two 1GB cards to test.. i tested both with the same USB/MicroSD adapter provided with the R4.. all of these tests were done at least twice, and were done after a fresh format on an empty card.. i was expecting there to be different 'actual size on disk' amounts between the FAT16 and FAT32 setups, but all 4 showed the same space on disk when i copied all my junk onto the card.. unfortunately the software doesn't seem to break apart between reading and writing, but rather just give an overall score for each file size..
San Disk [FAT16] - blue
San Disk [FAT32] - purple
Kingston [FAT16] - red
Kingston [FAT32] - green
you'll note that the San Disk (blue/16, purple/32) was basically the same between FAT16 and FAT32.. the Kingston (red/16, green/32) FAT16 destroyed all the other results, but the same card formated as FAT32 scored low.. i was actually quite shocked.. i tried each of these cards in loading/saving in animal crossing, and they all came out at the same time within a margin of error (i was using an online stopwatch).. in other words any differences in performance were negligible (with the 512kb save files at least)..
so, depending on your card it may be totally possible to have higher read/write speeds on FAT16 (or FAT32), but whether or not that will translate to anything when in use is a totally different question.. you'll notice both cards in both formats ended up with almost exactly the same stats at 2MB and above, so my guess is that any performance increases will be few and far between, and mostly with tiny files..