World number one Roger Federer suffered a shock 3-6 6-3 7-6 (7-4) defeat to world number 175 Thanasi Kokkinakis of Australia at the Miami Open.
The 36-year-old Swiss regained the top spot last month but will be replaced by Rafael Nadal in the new rankings.
He began in typically assured style but was worn down by qualifier Kokkinakis, 21, who won in two hours 21 minutes.
British number one Kyle Edmund lost 7-6 (7-4) 4-6 7-6 (7-5) to 20-year-old world number 63 Frances Tiafoe of USA.
Federer saw a 17-match unbeaten run come to an end in a gruelling match with Juan Martin del Potro in Indian Wells last weekend.
The 20-time Grand Slam champion was up against another angular opponent and endured a second loss in six days as Kokkinakis used a vicious top-spin forehand to devastating effect.
“It felt like every time I had chances something bad happened – wrong decision making by me, good decisions from him,” defending champion Federer said.
“It’s disappointing, I don’t know why I could never get to a level I was happy with.”
Meanwhile Edmund broke serve in the fifth game of the deciding set with Tiafoe, but was broken back by the American as he served for the match.
Tiafoe surged clear in the tie-break and, though the Briton saved five match points, he eventually succumbed to defeat in two hours 25 minutes.
Tiafoe won his first ATP Tour event at the Delray Beach Open last month, beating Germany’s Peter Gojowczyk in the final to become the youngest American to win a title since Andy Roddick in 2002.
In the men’s doubles, Britain’s Jamie Murray, seeded sixth with Brazilian partner Bruno Soares, advanced to the last 16.
The two-time Grand Slam winners recorded a 6-3 6-4 win over America’s French Open doubles winner Ryan Harrison and Max Mirnyi of Belarus.