I suppose this is a bit of a silly question but I just want to be sure: I clone my HD, which contains my vaults, nightly using SuperDuper.

Will the vaults work as expected in case I have to use the cloned disk?

Yes, provided that the vaults are closed during SuperDuper’s backup, so that the vault data will stay the same during the time it takes to copy it over to the destination.

Should you choose to back up vaults with Knox itself, the beta version of Knox 1.6 can now make open vaults read-only for the time of their backup, which will guarantee the same thing.

What about if they’re NOT closed but nothing’s done with them (e.g. because no one is using the computer at the time of the backup)?

Or do I absolutely have to close the vaults when I leave my desk in the evening for the backup to go through properly?

Spotlight might be indexing the volume during and end up with a broken index, which isn’t quite critical, but you’ll have to make sure that any application that stores data on the volume does not rely on tasks that fire up when the user is idle, and end up with partial data on the side of the backup.