Lukas Rosol could not maintain the stellar form that overcame Rafael Nadal and went down in straight sets to Germany's Philipp Kohlschreiber.
Rosol pulled off one of the biggest upsets in history when he knocked out the two-time champion on Centre Court, but lost 6-2 6-3 7-6 to Kohlschreiber.
Andy Roddick failed to make the second week for the second year running, losing to seventh seed David Ferrer.
Fifth seed Jo-Wilfried Tsonga went through with a straight-sets victory.
After his heroics on Centre Court, Rosol was sent out to Court 12 to face 27th seed Kohlschreiber.
But the Czech Republic's Rosol made 17 unforced errors and won only 69% of points on his first serve compared to figures of three and 83% respectively from Thursday's five-set thriller against Nadal.
"Of course, the atmosphere was really different, but still it cannot be every match with atmosphere like this two days ago," he said.
"Today was a little bit different conditions. It was windy. He had probably more power than me."
Rosol refused to dwell on his victory against Nadal but said he hoped it would send out a warning to future opponents.
"For me it's already past," he said. "I have to look forward, what's going to happen next days, next matches. I cannot live from this match that happened already. I have to live what's going to be.
"I can play with anyone. That's what I feel. And also these players now are going to see they can lose against me."
Kohlschreiber admitted the Wimbledon debutant was a serious threat despite losing in the first round of qualifying for the last five years.
"I think it was very dangerous to play against him," said Kohlschreiber. "But I think I have the right game plan against him. I mixed up the pace a lot. Every time if he hit a very strong ball, I tried to slice it back, keep it short. He doesn't like to move too much into the court.
"Everything I saw against Nadal, I figured out I think the perfect tactic. Obviously I played a very, very good match."
Roddick won the first set against Ferrer but the Spaniard battled back well to take the match 2-6 7-6 (10-8) 6-4 6-3.
But it was Roddick's farewell gesture to Centre Court which was the talking point.
The 29-year-old, who won the Eastbourne title the week before, applauded all four sides of the court, blowing a kiss to the fans.
"I don't have a definitive answer, I can't give you much else," said the three-time runner-up when asked if Saturday was his last Wimbledon.
Ferrer will now meet Juan Martin del Potro of Argentina, who brushed past Japan's Kei Nishikori in straight sets, winning 6-3 7-6 6-2.
It was a straight-sets victory for France's Tsonga also, beating unseeded Slovakian Lukas Lacko 6-4 6-3 6-3.
Tsonga will face American Mardy Fish in the last 16 after the American continued his progress with a 6-3 7-6 7-6 victory against Belgium's David Goffin.
Qualifier Brian Baker continued his remarkable run with a 6-4 4-6 6-1 6-3 win over France's Benoit Paire on Court Three.
The 27-year-old American only returned to professional tennis last July after almost six years out with a succession of injuries.
Baker went to college and took a job as a tennis coach, but he won a Challenger event in Florida to get a wildcard into the French Open, and then reached the final of his first ATP World Tour event for seven years in Nice before coming through Wimbledon qualifying.
"I've always been confident in my game. I always knew I was a good player," said Baker.
"It was just whether the body would cooperate and whether I could get more than even six, eight, 12 months healthy and able to play.