(CNN) -- It was deja vu for the Williams sisters at the French Open -- but not in a good way.
A mouthwatering third-round clash between seven-time grand slam champion Venus and 17-time major winner Serena was ruled out when the former lost to unseeded Slovakian Anna Schmiedlova 2-6 6-3 6-4 in early play in Paris on Wednesday.
Then hours later, Serena -- also the defending champion and world No. 1 -- fell to Spain's Garbine Muguruza 6-2 6-2, her worst ever grand slam performance.
In 2008, the siblings were also defeated on the same day at Roland Garros, their least productive major. The last time it happened was at Wimbledon in 2011.
An 18th grand slam singles crown for Serena would have tied her with legends Chris Evert and Martina Navratilova but the younger of the tennis playing sisters has now been upset in consecutive majors.
Venus has been hindered by debilitating autoimmune disease Sjogren's Syndrome in recent years though she looked solid in her first round match against the promising Swiss, Belinda Bencic.
The siblings join other big names who've already been upset at the tournament, including Australian Open champions Stan Wawrinka and Li Na, as well as Kei Nishikori and Caroline Wozniacki.
Defeat for Serena meant it was the first time in the Open era that the top two women's seeds had fallen before the third round.
"I don't think anything worked for me today," a dejected Serena told reporters at her post-match press conference.
"It was one of those days. You can't be on every day, and, gosh, I hate to be off during a grand slam but it happens. It's not the end of the world.
"I think she played really well and she played really smart. It's great because I'm going to go home and work five times as hard to make sure I never lose again.
"I feel like I don't have to win another match, I don't have to win another tournament. Everything and every day is a bonus for me.
"Obviously I want to do the best and I want to win and I want to be the best and that's my whole goal. But it's great sometimes to get knocked down because you have to get back up. I love getting back up. I love the challenge."
As for Muguruza, the 20-year-old played with precision and power, securing the biggest victory of her career to date with barely a hint of nerves.
"(Serena) said that if I continue playing like this, I can win the tournament. I said, 'I will try, I will try,'" Muguruza explained of her chat with her opponent at the net upon victory.
"I really didn't expect that but I'm really happy. I had to be aggressive and I did it well. Serena is a great champion but today is a great day for me."
There were no such problems for the No. 3 seed from Poland, Agnieszka Radwanska, who beat Czech Karolina Pliskova. Maria Sharapova -- the No. 7 seed from Russia -- disposed of Bulgaria's Tsvetana Pironkova 7-5 6-2
Angelique Kerber, the eighth seed from Germany, edged past American Varvara Lepchenko 6-2 7-5 while the 2011 U.S. Open champion Sam Stosur, from Australia, beat Austria's Yvonne Meusburger.
The No. 12 seed, Flavia Pennetta from Italy, was knocked out by Sweden's Johanna Larsson while there were also wins for Eugenie Bouchard, the 18th seed from Canada, and Spain's 14th seed Carla Suarez Navarro.
The top of the men's draw didn't see any such turmoil, and two of the favorites enjoyed straight sets victories on Wednesday.
Novak Djokovic, the No. 2 seed from Serbia, breezed past France's Jeremy Chardy 6-1 6-4 6-2 in just over 90 minutes. Djokovic is looking for his first French Open title and will now face Crotia's Marin Cilic.
Roger Federer, the 2009 champion, was also in cruise control as he dispatched Argentina's Diego Sebastian Schwartzman 6-3 6-4 6-4. The No. 4 seed will face Russia's Dmitry Tursunov in round three.
Both players sympathized with Stan Wawrinka, the Australian Open champion who was knocked out in the opening round by Spanish outsider Guillermo Garcia-Lopez.
"I understand what Wawrinka is going through. In 2008 when I won my first grand slam, I know how that period went after that," Djokovic told reporters.
"It's the first time that you have to encounter pressure or expectations of being a favorite in grand slams."
Federer also had some kind words for his compatriot, and said he was confident this would only prove a blip in his career.
"With Stan, I understand where he is, because I was in such a position at a moment in my career, and it's not even the pressure itself. I think it's how you look at yourself and at your game," the Swiss told reporters.
"Without changing everything, this is what he's got to work on and solve this, fix this. Little by little. It will not happen overnight.
"But with Australia, with the Monte Carlo Masters, it's in his pocket already. Nobody can take this from him. It will give him time."
Elsewhere on Wednesday, there were wins for the Czech No. 6 seed Tomas Berdych, Milos Raonic, seeded 8 from Canada, and France's No. 13 seed Jo Wilfried Tsonga.