For example, when you analyse Untreated condition vs Day1Treatment condition with only these samples in your design you will get some size factors.

If you have also set up a Day6Treatment condition in your design (but you don't use it yet), and want to to do the same comparison Untreated vs Day1Treatment, the size factors will change. (taking into account Day6)

I thought size factors was only relative to library size of its sample , so why do they change dependent on the design file even if you don't use a set of samples.

I was doing analyse Day1Treatment vs Untreated  & Day6Treatment vs Untreated with two separate design files.
But now I am wondering if it's better to have one design with all the samples, and do the two comparison to get same sizefactors because at the end of the day you finish with different differential genes detected.


SizeFactors are calculated based on all the samples in your dds object. So if you have 1 large experiment its better to put everything together and then perform comparisons for example using contrasts, rather than making new dds objects with subsets of your original dataset.