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World champions Dai Greene and Mo Farah found themselves overshadowed by British team-mate Perri Shakes-Drayton at Crystal Palace.
Shakes-Drayton starred in the Aviva London Grand Prix at Crystal Palace on Friday night.
Olympic team captain Greene had been hoping to break Kriss Akabusi's 20-year-old British record of 47.82 seconds in the 400 metres hurdles, but could only finish second behind Puerto Rico's Javier Culson in 48.10secs.
And although Farah enjoyed a comfortable victory over 5,000m, the performance of the night came from Shakes-Drayton in the women's 400m hurdles, with the Londoner storming to a surprise win in 53.77s.
Former Olympic champion Sally Gunnell is the only British athlete to have run faster and Shakes-Drayton's time was also joint second fastest in the world this year, taking 0.41s off her personal best set when winning European bronze in Barcelona two years ago.
European champion Irina Davydova of Russia was second and Jamaica's Kaliese Spencer third, with Beijing Olympic champion Melaine Walker more than 1.7s behind Shakes-Drayton in fourth.
The 23-year-old, who had been ranked just 21st before the race, said: "I'm over the moon with the time. I thought it was an opportunity to race the best girls which I haven't had this year.
"I thought 'Come on girl, pull your socks up, you have to go for it', and I sure went for it. I just believed in myself and reminded myself what I've done. I've had a good day at the office.
"It gives me confidence knowing that my training is going well but I'm not going to let it get to me too much. I'm still going to remain level-headed because it's not finished yet."
Greene had looked back to his best in Paris last week, clocking a new personal best of 47.84 to miss Akabusi's record by just 0.02s, but he again had to settle for second place behind Culson tonight.
"I wanted a bit more, I would have loved to get the win before a home crowd," the 26-year-old Welshman said.
"My top-end speed has been better recently but it needs to improve again and hopefully I can get it right for the Olympics.
"I think it was my third fastest time ever so I can't be too disappointed. I've got a few more weeks to get a bit sharper for the Olympics and I'm going in the right direction I think. I'm looking forward to the Olympics and hopefully I can get one over Culson then."
Britain's Jack Green was fourth in the same race, setting a new personal best of 48.60s, while Lawrence Okoye had earlier finished third in the discus with a best of 63.33m.
Farah waited until the last two laps of his final race before the Olympics to hit the front and gradually wind up the pace, eventually winning by almost four seconds in a time of 13:06.04.
"I'm in great shape," the 29-year-old said. "It was important to work on my speed a bit. Conditions were not great but I love this track and this crowd and I'm looking forward to the Olympics. It's not long to go.
"I just have to think of it as another race and forget it's the Olympics, but I'm quite looking forward to it."
American Tyson Gay won the 100m final in a time of 10.03s into a headwind of 1.2 metres per second, with compatriot Ryan Bailey and Jamaican Nesta Carter in second and third.
"I'm in pretty good shape, I just need to work on some things on my groin area with my physio and I should be good to go," the former world champion said.
"I feel I know how to mentally prepare for this type of weather and this was the best place to prepare for the Games."
British quartet Dwain Chambers, Mark Lewis-Francis, Andrew Robertson and Simeon Williamson had all failed to advance from the earlier heats, with Chambers the quickest in 10.35s.
Britain's Julia Bleasdale recorded a new personal best of 15:10.06 in the women's 5,000m, although she was a distant sixth as Vivian Cheruiyot led a Kenyan 1-2-3-4.
And there was disappointment for world silver medallist Hannah England in the 1,500m, who understandably trailed in last in her first race since May 27 when she suffered a spiked Achilles.
England, who spent five days in hospital and was banned from standing for more than three minutes at a time for two weeks, clocked 4:14.45 as Olympic team-mates Laura Weightman and Lisa Dobriskey were fourth and seventh respectively behind Bahrain's Maryam Jamal.
Andy Pozzi reached the final of the 110m hurdles but then pulled up at the third barrier as American Aries Merritt equalled his own fastest time in the world this year with victory in 12.93s. Former Olympic champion Liu Xiang had qualified for the final but withdrew with a back strain.
Britain's Chris Tomlinson recorded a season's best of 8.26m to finish second in the long jump, just two centimetres behind Australian Mitchell Watt.