This might be a basic question but I have a question on how to normalise a western blot when you have multiple controls.

I have some western blots using human samples and they go roughly like this:

Control, Control, disease 1, disease 1, disease 1, disease 2, disease 2, disease 2.

I have 2 controls on there, I want to normalise to HSP90 as a loading control, but as I have 2 control samples do I take the average of these? and then when I calculate fold change is it again from the average of the 2 normalised controls?

Thanks