Isner, a Greensboro, N.C. native and local favorite, cited a hip injury, while the Czech Republic's Berdych retired with right shoulder pain.
The absence of the third-seeded Isner breaks his recent string of successes in the run-up to the Wake Forest University event, including a title in Atlanta and a runner-up finish last week against world No. 1 Rafael Nadal at the ATP Masters 1000 event in Cincinnati.
The 6-foot-9 Isner was also a hard court runner-up in Washington this month.
He bested Berdych in the finals of last year's U.S. Open tune-up at Winston-Salem.
Notable deaths of 2014 [PHOTOS]
Michael Sam cut from the Dallas Cowboys