Born in Miami Beach, Florida, Middendorf was in her senior year at Pickens High School in Jasper, Georgia when, in 1987, she left to enter a performing arts school in Miami. She later attended the State University of New York (SUNY) at Purchase.
In 1992, she won the role of Carrie Brady on the long-running soap opera Days of Our Lives. The following year, she began playing the recurring role of Laura Kingman during the fourth season of Beverly Hills, 90210. In 1994, she made her film debut with a supporting role in Wes Craven's New Nightmare, co-starring Fran Bennett, Cully Fredricksen, and Matt Winston.
In the first ten years of her career, she was seen in such television dramas as Murder, She Wrote (in a 1996 episode with fellow DS9 guest-stars Richard Beymer, Meg Foster, and Richard Libertini), Touched by an Angel (in a 1997 episode written by Ken LaZebnik and co-starring Clive Revill), L.A. Doctors (in a 1998 episode with John Billingsley), Ally McBeal (two 1999 episodes, including one with Albert Hall and Ray Walston), Family Law (with Scott MacDonald, Christopher McDonald, and Julie Warner), Six Feet Under (with Ed Begley, Jr.), JAG (with Zach Grenier, Scott Lawrence, and Terry O'Quinn), and Any Day Now (with Daniel Dae Kim and Tom Wright). She also had a recurring role on The Practice, working with the likes of Bruce Davison, Bruce French, John Larroquette, Richard McGonagle, Lawrence Pressman, and Steve Rankin.
Middendorf's subsequent science fiction credits following her appearance on DS9 include an episode of Perversions of Science with Chris Sarandon, the first episode of Angel, in which she played the very first "damsel in distress" the good vampire tried to save in the series, and a 2000 episode of The X-Files with Randy Oglesby. Her more recent TV work include four episodes of 24 (co-starring Michelle Forbes) and guest spots on CSI, Cold Case (with Michael Nouri), Medical Investigation (starring Neal McDonough), House M.D., Without a Trace (co-starring Enrique Murciano and John de Lancie), and Shark (starring Jeri Ryan).
Middendorf has worked several times with J.J. Abrams over the years. She appeared in two episodes of Abrams' hit series in 2003, with Terry O'Quinn in the first and Christian Slater in both. The first of those were written by Alex Kurtzman and Roberto Orci and directed by the latter. Three years later, Middendorf appeared in Abrams' feature film directorial debut, Paramount's Mission: Impossible III (also co-written by Kurtzman & Orci and co-starring Simon Pegg). Most recently, Middendorf appeared in two episodes of Lost, the hit ABC series co-created by Abrams and Damon Lindelof and starring Daniel Dae Kim and Terry O'Quinn.