Thanks Marc, that is extremely useful. I should have remembered the essential "reset all, emphasis on ALL settings" before starting a new photo shoot! I had been out the day before shooting moving wildlife and had forgotten to restore AF-S.
I originally took the images in RAW and converted to jpeg. The problem with that was that the EXIF data that FocusTune needs was stripped out in the conversion. So, if I had uploaded the RAW images into FocusTune without any conversion there would be no picture controls to worry about. It isn't an issue anyway as I never shoot in JPEG when out on a shoot so no picture controls including contrast, sharpening etc are set in camera.
I will rerun the test tomorrow paying attention to ensure single point focus.