I realize this is an older post but I had the same error on IIS 8.5. Hopefully this can help another experiencing the same issue (I didn’t see my issue outlined in other questions with a similar title).
Everything seemed set up correctly with the Application Pool Identity, but I continued to receive the error. After much digging, there is a setting for the anonymous user to use the credentials of the application pool identity or a specific user. For whatever reason, mine was defaulted to a specific user. Altering the setting to the App Pool Identity fixed the issue for me.
- IIS Manager → Sites → Website
- Double click “Authentication”
- Select Anonymous Authentication
- From the Actions panel, select Edit
- Select Application pool Identity and click ok
Hopefully this saves someone else some time!