Swedish runner Sarah Lahti - who finished 12th behind Ayana - was dubious about the winner's preparation.
"I do not believe that she is one hundred per cent. It is too easy for her," Lahti told Swedish newspaper Expressen.
"One cannot see a facial expression, and she just runs on while others struggle for life behind her.
"I cannot say that she is not clean, but there is little doubt."
Questioned in the post-race press conference about the allegations, Ayana insisted she was clean, attributing her success to her lead-in, and to God.
"My doping is training. My doping is Jesus," Ayana said via a translator.
"Otherwise I'm crystal clean."
The race was marked by staggering pace, with 18 runners recording personal bests - including the first 13. Among them was Australian Eloise Wellings, who finished 10th.
Sonia O'Sullivan - the Irishwoman who won silver in the 5000m at the Sydney Olympics 16 years ago - was another who cast doubt over Ayana's achievement.
"That's a world record that many people would have questioned for a long time, 23 years ago set by the Chinese. I'm not jumping out of my seat because you question it as well yourself, how can you do that? It is 23 years later. Is it 23 years of knowing more training, being better athletes? How do you have an athlete that can break the world record so easily like that? She didn't look very tired afterwards.
"Things that you thought were impossible, were out of reach and would never be broken and then all of a sudden they are broken by the length of a straight.
"You do have to question it a little bit."
