Is "in" necessary in this sentence? What does refer to?
Excellent question. "Difference in" is usually used to refer to a change in one thing, instead of noting the dissimilarity between two things. In this case the thing is a list of things. So a "difference in how you feel", "a difference in how you think" etc. Ex: I noticed a difference in the way you play piano now. (How you play piano gas changed). Ex: There's a difference in my attitude. (My attitude has changed.) So to show that there is a change "in" is necessary.
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