Hi @Flemming,
This can certainly be the case, especially since different dynamic masks are generated and used for each refinement, so you can even see variance in the final resolution run-to-run with the same particles and initial volume because FSC calculation is mask sensitive. If each class showed a relatively minor conformational change, this could likely explain the 0.1 Å resolution different you saw. For larger differences between classes, it’s certainly true that the reference bias can exist. This can be reduced by tricks such as using a stronger initial low-pass filter.
Best,
Michael