No.10 seed Karolina Pliskova (CZE), pulled the ultimate upset by beating an injured Serena effectively ending her latest pursuit of history.

Williams, who double-faulted on match point, said her left knee, which she thought was injured during a second-round match, was a factor Thursday night at Arthur Ashe Stadium.

Williams’ coach, Patrick Mouratoglou said the injury obviously affected Williams on the court, and she tried to compensate by playing a riskier style than usual.

"At the end she tried to go for the winner on every shot," Mouratoglou said. "This with an injury. Even if she wins the second [set], it's not the point; she's not going to win the third. She cannot move. There is no match."

While Williams is known to have the best serve ever in women's tennis, it was second tier against Pliskova, who had seven aces and got 84 pct of her first-serve points.

Pliskova joined the club of only four singles players who had beaten Serena and Venus Williams in the same Grand Slam event. The others -- Martina Hingis, Kim Clijsters and Justine Henin -- all retired as decorated champions.

The incredible list of lifetime achievements Williams had within her reach -- an Open-era record 23 Grand Slam titles and an unprecedented seventh US Open championship -- will have to wait until next year.

The No. 1 ranking, which she has held for 186 consecutive weeks, will now go to Angelique Kerber when the new rankings are released Monday.