Isner was in-form in an opening round win against Federico Delbonis on Monday, a match where he never faced break point, but news of his withdrawal came late Wednesday. It's just the third walkover that Isner's given in his career, and first at a Grand Slam.
Teenager Rune, the No.28 seed, advances to Round 3 at the US Open for the first time as a result. He'll face seventh seed Cameron Norrie, who disposed Portuguese veteran Joao Sousa in straight sets on Thursday.
The 19-year-old Dane is playing in his second US Open, and fifth Grand Slam main draw. This spring, he reached the quarterfinals at Roland Garros.