All alignment tools require downtime while the VM is being alligned that is just the nature of the process being completed. Some streamline the process a bit but in the end downtime is still required.
The only trick that I can think MIGHT work is that the new converter for vsphere 5 can both align and sync changes so if you converted each VM back to the infrastructure and had it keep track and sync the changes it is POSSIBLE that you could severly reduce the downtime nessisary.
I would note this process has never been tested by me nor have i read anyone attempting this before so may want to testlab under real read write data scenarios
Best 3rd party tool for fixing VMDK alignment w/o VM down time