Osaka had not lost for over a year prior to Wednesday's outing, putting together the ninth-longest unbeaten run seen on the WTA Tour since the start of 2000.
However, this year's Australian Open champion was simply unable to match her opponent's high standards in their quarter-final clash, resulting in a 6-0 6-4 triumph for the excellent Sakkari.
World number 25 Sakkari had battled hard to get past Jessica Pegula in the previous round – including saving six match points – but needed just 69 minutes to see off four-time grand slam champion Osaka, who managed just five winners during her surprisingly brief stint out on court.
Afterwards, Sakkari revealed how coach Tom Hill helped devise a strategy to trouble the second seed – albeit she had to come from 3-0 down after a difficult start to the second set.
"I don't think tennis-wise it was like the best tennis I have ever played in my life," said Sakkari, according to the WTA Tour's website.
"I think I executed our strategy with Tom really well. I just did what I had to do. I'm not gonna tell you what. What we discussed before the match, I just did it most of the match, except maybe a couple of games where things didn't go right."
Osaka's previous loss was back in February 2020, when she was also beaten in straight sets by Sara Sorribes Tormo at the Billie Jean King Cup.
Since then, she has won last year's US Open and then the first grand slam in 2021, though the loss to Sakkari did not come as a complete surprise to her having struggled for rhythm in Miami.
"She's a really big fighter so I knew it wasn't going to be easy. I felt like I haven't been playing well this whole tournament," Osaka said.
"I couldn't find a groove, so mentally it's really hard for me to play against really high-quality players with what I feel is low-quality tennis."
Next up for Sakkari in the semi-finals is another former grand slam champion – Bianca Andreescu.
Triumphant at Flushing Meadows in 2019, eighth seed Andreescu outlasted Sorribes Tormo 6-4 3-6 6-3 in the day's final match at the WTA Premier event.
Osaka reached the last eight at the WTA Premier event for the first time in her career thanks to Monday's straight-sets win over Elise Mertens.
World number one Barty overcame two-time grand slam champion Victoria Azarenka in a tough three-set battle in Miami.
THERE'S NO STOPPING OSAKA
Australian Open champion Osaka extended her winning streak to a remarkable 23 matches.
Osaka defeated 16th seed Mertens 6-3 6-3 to stay hot – Venus Williams, Serena Williams, Justine Henin and Azarenka are the only other players with winning streaks of 23 or more matches since 2000.
"It definitely feels very special," four-time major winner Osaka said. "This is one of my favourite tournaments to play, and of course I'm sad I hadn't been able to get to the second week the last times that I have played this tournament. But I'm here now, and hopefully it will go well this time."
Maria Sakkari is next for Osaka after the 23rd seed saved six match points in her 6-4 2-6 7-6 (8-6) win over Jessica Pegula.
BARTY WINS BATTLE OF PAST AND PRESENT NUMBER ONES
In a rollercoaster battle, top seed Barty prevailed 6-1 1-6 6-2 against 14th seed Azarenka.
Barty extended her winning streak in Miami to nine matches after coming out on top against the former world number one.
"Playing against Vika you have to be ready right from the first point," Barty said. "I felt like I did that well in the first set and she gave me a few cheap errors. Then through the second she lifted her intensity.
"I probably wasn't sharp enough in those first couple of games to be able to go with her, particularly with new balls. When Vika is ahead and she's a front runner, she's the very best in the world."
Aryna Sabalenka stands in the way of Barty and the semi-finals after the seventh seed trounced Marketa Vondrousova 6-1 6-2.
SVITOLINA WINS THRILLER, ANDREESCU RALLIES
Fifth seed Elina Svitolina rallied past two-time Wimbledon winner Petra Kvitova 2-6 7-5 7-5.
Svitolina made a slow start against the red-hot ninth seed, but the 2018 WTA Finals champion finished strongly to progress.
Anastasija Sevastova took down Ana Konjuh 6-1 7-5 to set up a clash with Svitolina.
Meanwhile, eighth seed and 2019 US Open champion Bianca Andreescu came from behind to defeat two-time slam winner Garbine Muguruza 3-6 6-3 6-2.
Ostapenko won as a wildcard in 2021, and the world number 14 confirmed her place in this year's showdown by overcoming Camila Giorgi on Friday.
She is the first female player to reach back-to-back finals in Eastbourne since Caroline Wozniacki in 2017 and 2018.
The Latvian, who is also going to compete for the doubles title, prevailed 6-2 6-2 and will now go up against Kvitova. The pair have faced off eight times previously, with each player winning four matches.
It is Kvitova's first appearance in a final in 2022, with the former world number two – and two-time Wimbledon champion – having ended Beatriz Haddad Maia's winning streak.
Haddad Maia won in Birmingham last week and Nottingham the week before, but her run came to an end at 12 matches, with Kvitova triumphing 7-6 (7-5) 6-4.
"For me, a final after almost a year [without one] will be great, so I’m glad already," said Kvitova, who was a runner-up at Eastbourne in 2011 and last reached a final on grass in Birmingham four years ago.
"Jelena loves to play here, obviously, we saw it, she has a really great game for grass."
Meanwhile, at the Bad Homburg Open, 2019 US Open champion Bianca Andreescu reached her sixth career final courtesy of a walkover against Simona Halep.
The Romanian withdrew from the semi-final clash with a neck injury, which will worry the 2019 Wimbledon champion ahead of the season's third grand slam at the All England Club.
"I am sorry that I had to withdraw today before my semi-final match," Halep said in a statement.
"But unfortunately I woke up this morning with a blocked neck and this is not allowing me to perform to the best of my ability."
Andreescu will face Caroline Garcia, who saved a match point before going on to beat fellow Frenchwoman Alize Cornet 7-6 (11-9) 3-6 7-5 to reach an 11th tour-level showpiece.
The American beat Maryna Zanevska 6-3 7-6 (7-4) on Saturday, hitting 35 winners and 13 aces on the way to victory.
Parks has risen through the ranks in the last year, and explained how a laid back attitude has been the secret to her success in France.
"I think the key was just staying focused, and taking my time out there," Parks said after her win. "Every time I got ahead of myself, I would kind of lose points, so I would tell myself to slow down.
"This whole week, I'm kind of relaxed this week and just kept playing my game and doing me."
The 22-year-old will face Garcia on Sunday after she eased to a 6-2 6-2 win against Camila Osorio in just an hour and 12 minutes.
At the Thailand Open, Lesia Tsurenko will take on China's Zhu Lin in the final after the Ukrainian came past top seed Bianca Andreescu.
The Canadian retired hurt in the second set, though Tsurenko already led 7-5 4-0.
"The way [Andreescu was] playing every point, like she has an idea in every point, so of course she's an amazing player," Tsurenko said. "It was a big challenge for me to handle this, and I was just saying to myself that I have to keep fighting, and this is probably the main key that worked today for me."
Zhu defeated compatriot Xinyu Wang 6-2 6-4 to advance to Sunday's final.
Garcia, the world number 10, entered the contest off back-to-back losses for the first time since March, and Collins made it three in a row as she was just a little too good with both her serving and return game.
Collins won 63 per cent of her service points, with Garcia at 52 per cent, and she ended up securing five breaks in the match.
She will play Martina Trevisan in the second round after the Italian defeated Colombian qualifier Camila Osorio 6-3 6-4.
Neither player had an ace in the match, but the big differentiating factor was Trevisan's ability to win points off her second serve, converting 50 per cent of her chances while Osorio won only one of nine (11 per cent).
The only qualifier of the day to get a win was Louisa Chirico, who beat fellow American Alison Riske-Amritraj 1-6 7-5 7-6 (7-5).
Chirico, the world number 196, will be rewarded for her win with a showdown against world number four Paula Badosa.
Madison Keys had no issues dealing with Australian qualifier Ellen Perez 6-1 6-4 in just over an hour, and Coco Vandeweghe defeated Sofia Kenin 6-1 1-6 6-4.
In the last match of the night, Canada's Bianca Andreescu won a two-hour-and-40-minute battle against Russia's Liudmila Samsonova 7-6 (7-1) 4-6 6-2.
The reigning US Open champion exited the Madrid Open at the last-16 stage, going down in three sets to Anhelina Kalinina after struggling with an injury which she admitted had been "taking its toll".
Raducanu said in the aftermath of that 6-2 2-6 6-4 defeat that she would only have given herself a "five per cent chance" of advancing after suffering the injury, and expressed doubt over her ability to compete in Rome.
However, the 19-year-old now insists she will be able to cope on the clay in Italy, saying she needs to adapt to the intensity of top-level matches after suffering from several injury problems this year.
"I think it's just coming from a lot of intensity and overload" Raducanu said. "My back, I'm managing it.
"It's fine. But it's just trying to adapt again to the long matches, to the intensity. I think that all of the small sort of niggles I'm getting, they're all related and connected to each other, when something is overcompensating perhaps."
Raducanu's first assignment in Rome will see her face Canadian Andreescu, who also won the US Open as a teenager when she shocked Serena Williams with a straight-sets final victory in 2019.
That contest will mark the first head-to-head meeting between the two players, and Raducanu was looking forward to facing the former world number four as she highlighted the contrast between conditions in Rome and Madrid.
"Of course, we are both pretty good players," Raducanu said. "It's going to be a good match-up. She's a great athlete and obviously a champion. She's got a really good attitude. I think it's going to be interesting.
"I think here is completely opposite [to Madrid]. It's quite heavy and slow, so there's going to be a lot longer points. It will be interesting to see what the differences are. But I can already feel them on the court tennis-wise."
The Briton, who won the US Open in 2021, went down 6-3 3-6 6-2 in a genuine tug-of-war against the 31st-ranked Canadian.
Andreescu sent down seven aces for the match, including four in the first set along with 12 winners as she claimed an early lead.
Raducanu responded with greater aggression, securing the crucial break in the eighth game, before serving out to level the contest.
But Andreescu showed composure and quality in the big points in the final set, winning the final four games to round out the victory, sealing a second-round clash with seventh seed Maria Sakkari.
Andreescu finished with 32 winners compared to Raducanu's 20, while the Briton committed 40 unforced errors.
World number 42 Shelby Rogers edged out USA compatriot Sloane Stephens in just under two hours, winning 6-4 3-6 6-2. Rogers will take on second seed Aryna Sabalenka, who is arguably the tournament favourite after top seed Iga Swiatek's withdrawal, in the next round.
Rogers was one of five Americans to progress, including 2020 Australian Open champion Sofia Kenin who defeated Australia's Storm Hunter 6-0 7-6 (7-5).
World number 35 Amanda Anisimova retired down 7-6 (7-5) 5-2 to countrywoman Madison Brengle.
Leylah Fernandez, the player whom Raducanu beat in the 2021 US Open final, advanced into the second round with a 6-4 6-3 victory over Lesia Tsurenko in 88 minutes.
The world number 12 was trailing 6-2 2-1 when calling time on the match against Andreescu in a highly anticipated showdown between two of the past three US Open winners.
Raducanu initially left the court for treatment ahead of the seventh game of the opening set for a medical timeout and completed just four more games.
Speaking after her withdrawal, coming two weeks before the French Open, Raducanu explained she did not want to aggravate an injury sustained at last week's Madrid Open.
"I thought maybe taking one, two days off, it would go away because a lot of the other small niggles I've had, they've kind of gone away after taking, like, two days off," she said.
"Then I got here and I was training, but it just didn't seem to get better. I was training with some limitations. I wasn't moving really.
"I was just playing where I knew where the ball was coming, just staying in one corner. I think I must have underestimated the unpredictability of competition in a match.
"The last few weeks have been really positive. I've learned a lot about myself and my game has definitely improved on this surface but I need to make sure my back is fully right.
"I need to just keep on it. I don't want to play my next match with a feeling of limitation because I think that I learned my lesson from this week, when to push, when not to push."
Ons Jabeur joined Andreescu in the last 32 with a 6-0 7-6 (7-1) victory over Sorana Cirstea to keep her impressive run of form going.
The Tunisian won her first WTA 1000 title in Madrid last week and has now won seven successive main draw matches for the first time in her career
Elsewhere in Tuesday's action, American qualifier Lauren Davis pulled off a shock 6-2 6-3 win against number 11 seed Jelena Ostapenko.
Fellow Americans Jessica Pegula, Madison Brengle and Amanda Anisimova also advanced, beating Liudmila Samsonova, Marta Kostyuk and Tereza Martincova respectively.
Anisimova, who is now the player with the most wins in three sets so far in 2022 with nine, will face Belinda Bencic for a place in the last 16.
Ninth-seed Raducanu was the biggest name in action on Tuesday and had been hoping to rack up three successive WTA Tour victories for the first time since her incredible US Open success in September last year.
But Kalinina, who saw off Sloane Stephens and Garbine Muguruza in her two previous matches, got the better of the Brit in an entertaining 6-2 2-6 6-4 win.
Raducanu had not dropped a set in either of her first two outings in Madrid but Kalinina quickly put an end to that.
While the teenager responded well in the second, getting an important break to make it 3-1, Kalinina rallied again in what was a closer deciding set.
The pair traded breaks and then Kalinina got another to set her en route to victory at 5-4 – she had to save one more break point but did ultimately see off Raducanu to book a clash with Jil Teichmann in the last eight.
Teichmann beat Elena Rybakina earlier in the day, dispatching the 16th seed in impressively comfortable fashion as she won 6-3 6-1.
Progression from the last eight for Teichmann will see her reach the semi-final of a WTA 1000 event for the first time.
Sara Sorribes Tormo, the last Spaniard left in the draw, defeated Daria Kasatkina 6-4 1-6 6-3 in something of a rollercoaster ride to line up a quarter-final contest with 12th seed Jessica Pegula.
The American kept her focus despite opponent Bianca Andreescu needing a medical timeout and rain causing a 30-minute halt, eventually winning 7-5 6-1.
The Canadian claimed the first championship of her WTA Tour career in California last year, kickstarting an outstanding 2019 season.
Andreescu claimed the Rogers Cup and then triumphed at the US Open, before a knee problem at the WTA Finals dealt her the latest setback of an injury-plagued career.
The world number four has not played since and will not be able to make her season debut at the upcoming WTA Premier tournament either, she revealed on Saturday.
"As many of you know, I've been working through an injury I suffered last year during the WTA Finals," Andreescu said.
"It's been a long road to recovery and, while I was looking forward to getting back on court and defending my title at Indian Wells, unfortunately I'm still not 100 per cent.
"I would like to thank you all for your continued support and [for] sticking with me on this journey. Your kind words have been so uplifting.
"While I am disappointed that I am not competing next week in a tournament where I have such great memories, I am focused on my recovery and rehab and working hard to get back on the court as soon as possible."
Tunisian 13th seed Jabeur defeated the Canadian second seed 6-7 (5-7) 6-4 6-1 in two hours and 39 minutes on Thursday.
The come-from-behind triumph was the second time in two matches that the Tunisian has rallied from a set down to win after beating Daria Kasatkina in three.
Jabeur had twice been a break up in the opening set before Andreescu claimed it in an tiebreak.
The 26-year-old Tunisian, who made the Wimbledon quarter-finals this year, responded by breaking at 5-4 to win the second set.
After Andreescu had an injury scare after landing awkwardly on her left foot late in the second set, Jabeur dominated the third, finishing by winning eight of the final nine games.
Jabeur finished with 9-3 aces and was more effective on serve, going at an 81.6 win percentage on her first serve (40 from 49 points).
The lower side of the draw has opened up for the Tunisian who will face Jessica Pegula in the quarter-finals after the unseeded American defeated countrywoman Danielle Collins 6-4 3-6 7-5.
Two-time Wimbledon champion and seventh seed Petra Kvitova was knocked out in a shock by Italian Camila Giorgi in straight sets.
Giorgi, ranked 71st in the world, won 6-4 6-4 in one hour and 36 minutes and will face Cori Gauff in the quarter-finals after she had another walkover against Johanna Konta.
Top seed Aryna Sabalenka had no such problems, cruising past Canadian Rebecca Marino 6-1 6-3 inside an hour.
Sabalenka sets up a quarter-final clash with fellow Belarussian and two-time Australian Open winner Victoria Azarenka.
Azarenka got past Greek 11th seed Maria Sakkari in three sets, 6-4 3-6 7-6 (7-2).
Fourth seed Karolina Pliskova got past Amanda Anisimova 6-1 7-6 (10-8) and will play Sara Sorribes Tormo in the last eight after she won in three sets over Katerina Siniakova.
Rybakina, 23, has been one of the most in-form talents in the sport this year, with her only two losses since the start of the Australian Open coming in three-setters against Aryna Sabalenka and Beatriz Haddad Maia.
The Kazakhstan representative produced 10 aces against Mertens on her way into the quarter-finals, becoming the first player this WTA season to finish with at least 10 aces in three consecutive matches.
She will look to keep her sparkling form alive when she meets Martina Trevisan for a spot in the semi-finals, after the Italian got the better of Latvia's Jelena Ostapenko 6-3 6-3.
Sabalenka showed why she is the top remaining seed after Iga Swiatek's withdrawal, beating 16th seed Barbora Krejcikova 6-3 6-2. Krejcikova was one of two players this season to beat Sabalenka – with Rybakina the other – and by avenging that defeat the Belarusian improved her record in 2023 to 20-2.
America's top hope Jessica Pegula needed only 79 minutes to eliminate Magda Linette 6-1 7-5, winning the first five games of the match to set the tone early, and she will now meet Russia's Anastasia Potapova in the quarters.
Potapova finished with just one ace compared to Qinwen Zheng's nine, but she showed enough guile to overcome the Chinese international's power advantage.
Romania's Sorana Cirstea kept her great run going with a 7-6 (7-3) 6-4 triumph over Marketa Vondrousova, making it eight wins from her past nine matches, and Bianca Andreescu was forced to retire through injury while down a set 7-6 (7-0) 0-2 against Ekaterina Alexandrova.
Sabalenka hit 26 winners in the victory and now has 250 at WTA 1000 level for the season, representing one of only two female players with more than 200 in 2023.
The world number two, who is the top seed in the tournament after Iga Swiatek's withdrawal, has now won 19 of her past 21 matches, with her only losses in that span to Elena Rybakina and Barbora Krejcikova.
She will get the chance to avenge that loss to Krejcikova next after the Czech Republic representative knocked out hometown star Madison Keys 7-6 (7-4) 6-3.
Former French Open winner Krejcikova registered her 10th WTA 1000 level win against Keys, becoming only the second player to achieve that this season.
Former Australian Open champion Sofia Kenin fell 6-4 6-4 to Canada's Bianca Andreescu, who continued her strong run after previously knocking out seventh seed Maria Sakkari and 2021 US Open champion Emma Raducanu.
In a serve-dominated match, 2019 US Open champion Andreescu sent down seven aces, maintaining 70 per cent first serve percentage.
Ninth seed and former Olympic gold medallist Belinda Bencic was eliminated 7-6 (10-8) 6-3 by 18th seed Ekaterina Alexandrova, who will take on Andreescu in the next round.
Marketa Vondrousova won her all-Czech matchup against 17th seed Karolina Pliskova, and their compatriot Petra Kvitova enjoyed a 6-4 7-6 (7-3) triumph over recent Monterrey Open champion Donna Vekic.
Romania's Sorana Cirstea made it seven wins from her past eight matches with a 7-5 6-1 result over Karolina Muchova, having knocked off fifth seed Caroline Garcia in the second round.
Varvara Gracheva ensured a strong day for the Russians with a 6-1 6-2 demolition of Magdalena Frech in a battle of two unseeded players.
The number two seed won 7-6 (10-8) 7-6 (7-3), saving seven set points after going 5-1 down in the first set, before roaring back to take both tie-breaks.
Sabalenka will face Marketa Vondrousova in the quarter-finals, with the Czech having produced a dominant performance in a 6-0 6-4 win against Kaia Kanepi.
"When it's 1-5 down, it's like you have nothing to lose, and you just go for your shots without thinking," Sabalenka said.
"That really helped me to stay in the set, to keep fighting and to keep trying."
Fourth seed Veronika Kudermetova eased through after a 6-4 6-0 victory against Bianca Andreescu, while Marta Kostyuk also advanced with a hard-fought 6-7 (5-7) 6-2 6-3 win over Elena Rybakina.
At the ASB Classic in Auckland, second seed Sloane Stephens is out after falling to Rebeka Masarova.
Having been forced to resume on Wednesday after rain suspended play the previous day, Stephens struggled against her Spanish opponent, eventually losing 6-3 7-6 (7-5).
Fifth seed Wang Xiyu is also out after retiring from her match against Karolina Muchova, but seventh seed Danka Kovinic is through after a simple 6-1 6-4 win against Nao Hibino.
Kovinic will face Lauren Davis next after she overcame Tamara Zidansek 6-2 6-1, while fellow American Sofia Kenin will take on number one seed Coco Gauff in the last 16 after she beat Wang Xinyu in straight sets.
A 6-4 1-6 7-5 victory for Madison Keys over top seed Sabalenka capped a day of upsets in Berlin.
American Keys fought back from a set down to bundle the world number four out in an hour and 29 minutes.
The battling Sabalenka saved 10 break points but was unable to avoid an early exit in the inaugural grass-court event.
Second seed Svitolina started her grass-court season with a 6-4 7-5 defeat to Ekaterina Alexandrova.
Russian Alexandrova fended off seven set points in the second set before sending the world number six, who was given a first-round bye, packing.
Svitolina, playing her first match since crashing out of the French Open in the third round, became the fourth top-10 scalp for Alexandrova, who will face Belinda Bencic in her fifth quarter-final of the year.
Andreescu's first match on grass since Wimbledon qualifying three years ago ended in a 7-6 (7-2) 7-5 loss to Alize Cornet.
The third seed from Canada parted with her long-time coach Sylvain Bruneau after she was beaten by Tamara Zidansek in the first round at Roland Garros and was unable to hit the ground running on grass.
Bencic, the fifth seed, advanced with a 6-3 5-4 success over Petra Martic.
Williams, playing for the first time since declaring on Tuesday her intention to retire after this month's US Open, was no match for Tokyo Olympics gold medalist Bencic.
The 23-time major winner was unable to claim back-to-back WTA singles wins for the first time since last year's French Open, having defeated Nuria Parrizas-Diaz for her first victory in 430 days on Monday.
Bencic triumphed in one hour and 17 minutes, winning 84.2 per cent of first-serve points and converted five of eight break points throughout the match.
Williams' power was on show with 13 winners, but Bencic was physically more capable and decisively managed 25 winners with only 13 unforced errors.
Elsewhere, 2019 US Open winner and local hope Bianca Andreescu edged Alize Cornet in a see-sawing clash in the evening, winning 6-3 4-6 6-3 in two hours and 26 minutes.
Fourth seed Paula Badosa and fifth seed Ons Jabeur, who was last month's Wimbledon runner-up, were forced to retire due to injury.
Spanish 24-year-old Badosa withdrew against Yulia Putintseva 7-5 1-0 due to muscle cramping, while Zheng Qinwen had a walkover against Jabeur 6-1 2-1 due to abdominal pain.
Top seed Iga Swiatek brushed aside Ajla Tomljanovic 6-1 6-2 in 64 minutes. The win means Swiatek is the first player to win 15 WTA 1000 matches in straight sets in a row since 2009.
Canadian 13th seed Leylah Fernandez also bowed out, going down 7-6 (7-4) 6-1 to Brazil's Beatriz Haddad Maia, who will face Swiatek in the third round.
Second seed Anett Kontaveit lost 6-4 6-4 to Jil Teichmann in one hour and 27 minutes. Teichmann will next face Simona Halep who won in 71 minutes against Zhang Shuai 6-4 6-2.
Sixth seed Aryna Sabalenka got past Sara Sorribes Tormo 6-4 6-3 to set up a third-round meeting with Coco Gauff after she defeated Wimbledon champion Elena Rybakina 6-4 6-7 (8-10) 7-6 (7-3) in an epic that lasted two hours and 49 minutes.
Jessica Pegula won 6-2 7-5 over American qualifier Asia Muhammad to progress through to face Camila Giorgi after she knocked off Elise Mertens 7-3 7-5.
Third seed Maria Sakkari triumphed in three sets 6-2 4-6 6-2 over Sloane Stephens and will face Karolina Pliskova next after the Czech beat Amanda Anisimova 6-1 6-1.
Eighth seed Garbine Muguruza won 6-4 6-4 against Kaia Kanepi and Alison Riske toppled 16th seed Jelena Ostapenko 7-6 (7-2) 0-6 7-5.
The US Open semi-finalist entered the WTA Tour event with the intention of gaining match practice on clay before the upcoming French Open.
That delayed grand slam takes place from September 27 to October 11 in Paris, switched from its original May start due to the COVID-19 crisis.
Williams fell short in her bid to land a record-equalling 24th grand slam when she was edged out by Victoria Azarenka in New York this week, when she needed medical assistance for the Achilles pain.
She stressed afterwards that she intended to play the French Open, but the Rome event, which begins on Monday, has now been scratched from her plans.
Former world number one Williams said in a statement released by the WTA: "I regretfully must withdraw from the Internazionali BNL d'Italia due to an Achilles strain."
Petra Kvitova and Bianca Andreescu will also be absent from the tournament after the WTA announced their withdrawals.
Simona Halep is among those returning to action, however, with the world number two having opted to miss the US Open due to concerns related to the pandemic.
The world number five lost 6-4 7-6 (7-3) to 21st-ranked Rybakina, whose reward is a meeting with Coco Gauff's conqueror Anastasija Sevastova in the quarter-finals.
Third seed Bianca Andreescu of Canada also lost in straight sets, Anett Kontaveit of Estonia beating her 6-3 6-3.
And it was the same story for fourth seed Iga Swiatek, though she at least took a set as she was beaten by Daria Kasatkina.
It took an impressive rally from Russia's Kasatkina to bounce back from losing the first en route to a 4-6 6-0 6-1 success.
She will now face Jelena Ostapenko, who beat Ons Jabeur 5-7 6-4 6-3.
In fact, top seed Aryna Sabalenka was the only seed to avoid a surprise exit on Wednesday as she cruised through.
The Belarussian, ranked fourth in the world, beat Alison Riske 6-1 6-4 in just over an hour to set up a clash with Camila Giorgi.
Giorgi had earlier followed up a win over defending champion Karolina Pliskova in the last round by beating Shelby Rogers 6-3 4-6 6-2.
There was no such string of shocks at Wednesday's other WTA event, the Bad Homburg Open in Germany, though first and second seeds Petra Kvitova and Victoria Azarenka needed three sets to progress.
Kvitova lost a second-set tie-break as she beat Ann Li 6-2 6-7 (4-7) 6-1 to reach the quarters, while Azarenka defeated Alize Cornet 6-4 3-6 7-6 (9-7).
Angelique Kerber, the fourth seed, progressed more smoothly, earning a comfortable 6-0 6-2 win over Russia's Anna Blinkova.
And Nadia Podoroska saw off the challenge of Patricia Maria Tig, winning 7-6 (7-4) 2-6 6-4.
The 2022 French Open and US Open champion triumphed 6-4 4-6 6-1 in two hours and two minutes, bouncing back strongly after losing the second set.
Zheng's power and forehand top spin proved challenging for the Pole, who was solid from the baseline to secure her 11st tour quarter-finals appearance this season.
The Chinese had broken Swiatek to lead 1-0 in the third set, but the top seed responded in trademark style, improving her record in the United States this season to 21-1.
Swiatek will take on eighth seed Coco Gauff in the quarter-finals, with the match-up marking a re-match of this year's French Open final.
Gauff worked her way into the last eight with a 6-4 4-6 6-3 victory over 2019 US Open champion Bianca Andreescu.
The 18-year-old American also had to fight back from a set down in the third set to win in a match that lasted just over two-and-a-half hours.
Third seed Aryna Sabalenka fought back from a lopsided opening set to knock out American Sloane Stephens 1-6 6-3 6-2 in two hours and one minute. The Belarussian will take on Donna Vekic in the next round.
Second seed Paula Badosa eased into the last eight, winning in 53 minutes against American qualifier Louisa Chirico 6-0 6-3. Badosa will face Danielle Collins in the quarter-finals.
Madison Keys triumphed over eighth seed Daria Kasatkina 6-4 6-3 in one hour and 27 minutes to secure a quarter-final date with fourth seed Jessica Pegula.
Swiatek, who became the first female player to win 25 consecutive matches since Serena Williams in 2015, will face Bianca Andreescu in the last eight after she eased past Petra Martic 6-4 6-4.
"I didn't start well, and everybody could see that," Swiatek told reporters. "I'm really happy with the way I reacted and how I improved in the first set.
"Also how different the second set looked to the first one because I could really reset and really change the way I played. That's the most positive thing for me."
There was a shock in the final game of the day, though, as Daria Kasatkina dumped out number two seed Paula Badosa 6-4 6-4.
Third seed Aryna Sabalenka sealed a third straight win over Jessica Pegula, easing to a 6-1 6-4 victory, while fourth seed Maria Sakkari defeated Coco Gauff 6-4 7-5.
Seventh seed Danielle Collins was knocked out by unseeded compatriot Amanda Anisimova, the 20-year-old cruising past the Australian Open finalist 6-2 6-2.
There were also victories for Jil Teichmann, who overcame Elena Rybakina 6-7 (3-7) 6-3 7-5, and ninth seed Ons Jabeur, who beat Yulia Putintseva 6-3 6-2.
