The 13th-seeded American retired with a nagging right ankle injury during his first-round match at the Australian Open on Tuesday after losing the first two sets to Slovakia's Martin Klizan 6-2, 7-6 (6).
Last year, Isner was forced to withdraw before the Australian Open even began with bone bruising in his right knee.
Both injuries happened in the same unlucky place for the American—the Hopman Cup in Perth, a team exhibition event that is one of the warm-up tournaments for the first Grand Slam of the season.
"I know movement is not the best part of my game," the 2.08-meter (6-foot-10) Isner said, "but at a certain point I've got to be able to move without pain and I wasn't able to do that today."
Isner was the only American man seeded at this year's Australian Open. (Canada, by contrast, has two—Milos Raonic and Vasek Pospisil—both of whom won their first-round matches.)
The big-serving Isner has struggled with injuries to his knees over the past year, retiring from his second-round match at Wimbledon with a left-knee injury after pulling out of the Australian Open with the right knee injury.
But he was feeling confident coming into the new season, largely because his knees felt fine.
He said after he tweaked the ankle in Perth, he decided to play another warm-up tournament in Auckland, New Zealand, last week to see if it would feel better.
"I was able to win my first match and didn't feel good afterwards and then I won again. The whole thing in Auckland was bizarre because I never expected to win that tournament," he said.
"I didn't come down here to win Auckland. I love that tournament but I came down here to try to do well here and that wasn't the case."
Isner said the injury isn't serious—he had an MRI in Perth and it didn't reveal any bone fractures.
He's returning to the U.S. to rest in a bid to be ready for a Davis Cup match against the Andy Murray-led British team in early February, which will be played at a makeshift clay court set up in the outfield at Petco Park, a baseball stadium in San Diego, California.
"I feel like I will get over (this injury). It doesn't concern me as much as some knee issues I've had in the past," he said. "I fully intend to get ready for Davis Cup."