Once Upon a Studio is a nostalgic tribute to Disney's animation legacy, featuring a vast ensemble of characters for a group photo. The film does not include any explicit or implicit LGBTQ+ representation or thematic focus, concentrating instead on celebrating the studio's centennial and character interactions.

Once Upon a Studio does not feature any identifiable transsexual characters or themes within its narrative. The film's focus is on a nostalgic celebration of Disney's animation legacy, rather than exploring specific social identities or communities.

The movie does not contain any action or adventure elements.

The film features a large ensemble of Disney characters, all of whom are portrayed with their original, canonical genders as established in their source materials. There are no documented instances of characters whose on-screen gender differs from their established gender, thus no gender swaps occur.

The film primarily features animated characters reprised by their original or archival voice actors, maintaining their established designs and portrayals from prior source material. New live-action characters are original to the film, not race swaps of existing roles.