Defending Paralympic champions Dave Roberts and Liz Johnson failed to hit the London 2012 qualifying times at the trials in Sheffield.
However, selectors can still pick them if they think they could win a medal.
Roberts, an 11-time gold medallist, missed both the 50m and 100m freestyle times, while Johnson, who won 100m breaststroke gold in her SB6 category in Beijing, failed to hit her mark.
"It's going to mean a tense few days for us," Johnson told BBC Sport.
European silver medallist Charlotte Henshaw also missed the standard.
Johnson and Henshaw both needed to reach the one minute 40.87 second standard but Johnson's best was 1:42.16 while Henshaw managed a best of 1:42.42 - the second and third-fastest times in the world this year.
"The only way you can guarantee selection is by reaching the qualifying times but if [performance director] John Atkinson thinks Charlotte and I should be on the team, I trust he will put us on the team," Johnson said.
"We also came close to reaching the standard at the first trials in London but it is a very competitive category and the qualifying is tough but if you set the bar that high you will get some great performances."
Roberts, who had only three weeks training coming into the trials after a run of illness and injury, accepted that he may have raced his final race.
The Welshman needs one gold medal to beat Baroness Tanni Grey-Thompson's Paralympic tally and become the most successful British Paralympian of the modern era.
"I tore my triceps last year and the surgeon said it should be a career-ending injury and if it wasn't for the London Games I probably would have retired," he said after missing the 100m qualifying time by just under two seconds.
"I couldn't have done much more but that last race was maybe one race too many.
"London is a home Games and I dearly want to be there but sometimes you have to listen to your body and maybe it is time to take stock."
The latest name to put themselves in contention for a place on the team is James O'Shea who was discovered by Olympian Mark Foster and who, at the age of 34, reached the qualifying standard in the 100m breaststroke in his SB5 category.