Maybe it was the daunting deficit Serena Williams faced in the French Open's third round. Maybe it was the way her 20-year-old American opponent, Sofia Kenin, was questioning line calls.

Either way, as Williams attempted to start a comeback Saturday with a three-ace game, she followed those big serves with some serious stare downs.

Whether it was meant to get herself going or intimidate Kenin, it didn't work. Outplayed from start to finish, Williams lost 6-2, 7-5 to the 35th-ranked Kenin, ending her latest bid for a 24th Grand Slam title with her earliest loss at a major tournament in five years.

Get Breaking News Delivered to Your Inbox

"In that first set in particular, she hit pretty much inches from the line, and I haven't played anyone like that in a long time," the 37-year-old Williams said. "I just saw a player that was playing unbelievable."

Serena Williams of The United States leaves the court following defeat during her ladies singles third round match against Sofia Kenin of The United States during Day seven of the 2019 French Open at Roland Garros on June 01, 2019 in Paris, France. / Getty Images

It was the second significant surprise in a matter of hours: Earlier in the day, No. 1 seed Naomi Osaka was eliminated 6-4, 6-2 by 42nd-ranked Katerina Siniakova of the Czech Republic. That ended Osaka's 16-match Grand Slam winning streak, which included titles at the U.S. Open final in September — when she beat Williams in the final — and at the Australian Open in January.

Osaka was trying to become the first woman to win three consecutive major trophies since Williams grabbed four in a row in 2014-15, a run that was preceded by a second-round loss at Roland Garros and a third-round loss at Wimbledon.

Since those early-for-her defeats, Williams had won six of the 14 majors she entered to surpass Steffi Graf's professional-era record of 22 Grand Slam singles championships. With 23, Williams stands one away from Margaret Court's mark for the most in tennis history; Court played in both the professional and amateur eras.

"Serena is such a tough player. I'm still trying to process what just happened," Kenin said, about an hour after the match ended with her covering her face with both hands. "She's a true champion and an inspiration."

Kenin was born in Moscow and is fluent in Russian. Her family moved to New York when she was a baby, and she now is based in Florida.

"I'm proud to be an American," said Kenin, who wore a blue U.S. Fed Cup cap to her news conference. "I think it's great we moved to America for a better life for me."

She is appearing in the ninth major of her career and now is headed to her initial trip to the round of 16, where she'll meet No. 8 seed Ash Barty. Other women's fourth-round matchups established Saturday: defending champion Simona Halep vs. 18-year-old Iga Swiatek of Poland; No. 14 Madison Keys of the U.S. vs. Sinikiakova; 17-year-old Amanda Anisimova of the U.S. vs. qualifier Aliona Bolsova of Spain.