Venus Williams and Kim Clijsters both fell at the first hurdle as seeded players dominated the women's draw for a second straight day at the US Open.
Hours after younger sister Serena secured her place in the second round, Venus, 40, was back in the spotlight in what was an incredible 22nd US Open appearance.
But Venus, who reached the final of the US Open on her debut at the tournament in 1997, was beaten in the first round for the first time in her glittering career by Karolina Muchova of the Czech Republic.
Muchova, the 20th seed, had too much poise for Williams en route to a 6-3, 7-5 win.
Defeat to the 20th seed means Venus has failed to go beyond the third round of a grand slam since 2017.
Despite her 6-3, 7-5 setback, the seven-time major winner insists that she plans to continue playing, starting with this month's French Open at Roland Garros.
"I love this game," Williams said. "I'm good at what I do. It's easy to stay motivated and excited about doing something so amazing. Not many people get to do this.
"But I look forward to a few days off. It's been a lot of work. Looking forward to just having a little bit of a rest before the clay courts."
Belgium's Clijsters meanwhile, making the second comeback of her career and her first appearance at a Grand Slam since 2012, raised hopes of an upset after taking the first set against Russia's Ekaterina Alexandrova.
The 37-year-old three-time US Open champion ran out of steam though as Alexandrova, seeded 21, completed a 3-6, 7-5, 6-1 win.
This year's women's field in New York is more open than ever with world number one Ashleigh Barty, the second-ranked Simona Halep and defending champion Bianca Andreescu all electing to skip the tournament.
One of the leading contenders for the title, second seed Sofia Kenin of the United States, got off the mark with a 6-2, 6-2 win over unseeded Yanina Wickmayer of Belgium.
The 21-year-old, who won a maiden Grand Slam title at this year's Australian Open, later revealed she had cried to herself before the match in order to alleviate stress.
"Crying. That's what I did. I mean, I had to let it out," Kenin said. "It's fine. It worked."
Kenin will play Canada's Leylah Fernandez in the second round. Fernandez opened her campaign with a 6-4, 7-5, win over Vera Zvonareva on Tuesday.
Ninth seed Jo Konta meanwhile won the 'Battle of Britain' against compatriot Heather Watson, winning 7-6, 6-1.
"There's always going to be rivalry between compatriots," Konta said. "It's always going to be that extra stressful.
"There's always going to be an element of people having more judgments and more of an opinion on the outside at home on a match like today," added Konta, who will play Romania's Sorana Cirstea in the second round.