I have a file that contains a set of Timestamps in format of H:M:S.MS, I can read and print all of the saved Timestamps but when I do some arithmetic operation
H:M:S.MS
Assuming your variables are integers, you need to enclose your calculation in an arithmetic evaluation for that to work.
echo $(( VAR1 - VAR2 ))
Or, if you want to assign the value :
VAR3=$(( VAR1 - VAR2 ))