Five-times champion Williams had cruised through the draw in Melbourne but was a shadow of her power-packed self against Russia's world number 56 Ekaterina Makarova, going down 6-2 6-3 in sweltering conditions at Rod Laver Arena.
A "bored" Andy Murray hurried past Mikhail Kukushkin after the Kazakh retired in the third set and will play Kei Nishikori in the last eight after the Japanese stunned world number six Jo-Wilfried Tsonga 2-6 6-2 6-1 3-6 6-3.
Williams plans to take a course in kinesiology, the study of human movement, when she returns to the United States and the 30-year-old looked in dire need of some instruction after a lackluster display.
The 13-times grand slam champion, who was the last American standing at Melbourne Park, was unable to defend her 2009 and 2010 titles last year due to a freak foot injury and had carried a sprained ankle into the tournament this year.
"Obviously I'm not 100 percent, and I haven't been. But it's no excuse or anything," a dejected Williams said. "I know that I can play 100 times better than I did this whole tournament.
"She went for broke on a lot of her shots. I made 37 errors. That kind of tells the story of the match."
LOST FOR WORDS
Makarova was lost for words after the stunning upset.
"It's an unbelievable feeling, I don't know what to say," she beamed in a court-side interview after sealing a spot in a grand slam quarter-final for the first time.
Czech Kvitova also booked her place in the last eight with a 6-2 7-6 win over Serbian Ana Ivanovic but the main talking point of the Wimbledon champion's win was a mis-timed smash that saw her completely miss the ball.
The world number two shrugged off the embarrassment of what may have been the worst shot of her career.
"I know that it looked really strange when I missed it," said the 21-year-old. "But anyway ... I won, that's the important thing."
In the men's draw, Murray was leading 6-1 6-1 1-0 when Kukushkin retired due to a hip injury. Murray had toiled against the Kazakh in the Brisbane warm-up tournament earlier this month but wrapped up Monday's win in just 49 minutes.
"Boring" was how the Scot described the game.
"I thought it was best he retired because it was pointless. The people probably weren't enjoying the match that much. I certainly wasn't because nothing was happening."
Murray's reward for the win was a quarter-final match against Nishikori, who came from a set down against Tsonga to become the first Japanese man to advance to the quarter-finals of the Australian Open in 80 years.
The 24th seed has now matched the feats of Ryosuki Nunoi and Jiro Satoh when they made the last eight in Australia in 1932.
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