I'm a grad student in behavioural neuroscience.

I am looking at individual difference in anxiety in animals.

There are two groups of animals: one anxious and the other non-anxious.

I present then with a potential threat and see if their anxiety levels are reflected on their behaviour.

In the test paradigm, I place a threatening object in front of a cage for a certain time.

The cage is divided into 4 sections: Front, Middle, Back and Furthest back.

I measure proportions of time spent at each location.

For instance, a particular animal spent 20% Front, 40% Middle, 30% Back and 10% Furthest back.

Thus, independent variable is the group; anxious and non-anxious.

Dependent variable is the proportion of time spent at each location.

In order to see if there is any difference between the groups, I am doing a t-test for each location.

But, would it be possible to do a 2-way ANOVA with independent variables: Group (Anxious, Non-anxious) and Location (Front, Middle) and dependent variable: proportion of time spent at each location?

Since the locations are not independent from each other, will this violate the independence of observation?

Thank you,