I have a CPT (defined in functions.php) that I have set to as non-hierarchical. A lot of content has already been uploaded into the database.
If I change the definition to 'hierarchical' => true, will the change affect any of the posts in that CPT?
No, it will not. It changes just the interface in the post editor: you get the page attributes box to select a parent.
All existing post have already a value for the parent:
0. You can find it in thepost_parentproperty of each post. The only thing that changes now is the ability to change that value.