The ad also features footage from the 1990s of Trump saying in an interview that Machado went from 117 or 118 pounds to 160 or 170 and commenting: "So this is somebody that likes to eat."
During an interview on Fox News on Tuesday morning, Trump brought up Machado on his own and launched into an attack on her credibility, saying that she had "attitude" and was a "real problem" for Miss Universe officials.
"She was the worst we ever had. The worst. The absolute worst. She was impossible," Trump said. "... She was the winner, and she gained a massive amount of weight, and it was a real problem. We had a real problem."
Trump continued: "Hillary went back into the years and she found this girl — this was many years ago — and found the girl and talked about her like she was Mother Teresa. And it wasn't quite that way. But that's okay. Hillary has to do what she has to do."
In the final minutes of the debate, Trump doubled-down on his assertion that Clinton doesn't look presidential or have the "stamina" for the job. Clinton used that as an opportunity to list unflattering things Trump has said about women.
"This is a man who has called women pigs, slobs and dogs, and someone who has said pregnancy is an inconvenience to employers," Clinton said, as Trump shouted out that he never said those things, "who has said women don't deserve equal pay unless they do as good a job as men. And one of the worst things he said was about a woman in a beauty contest. He loves beauty contests, supporting them and hanging around them. And he called this woman 'Miss Piggy.' Then he called her 'Miss Housekeeping,' because she was Latina. Donald, she has a name."
"Where did you find this? Where did you find this?" Trump demanded.
"Her name is Alicia Machado," Clinton said.
Trump responded by saying that while he has said "very tough things" to Rosie O'Donnell, "I think everybody would agree that she deserves it and nobody feels sorry for her." He also pointed out that he could be "extremely rough to Hillary, to her family" but would hold himself back from doing so.
On Fox News on Tuesday, Trump clarified that he refrained from bringing up "all of the women" that Clinton's husband, former president Bill Clinton, has had affairs with.
"I didn't feel comfortable doing it. I think I did the right thing," Trump said. "It's not worth a point. I didn't feel comfortable doing it with Chelsea in the room — I think Chelsea is a fine young woman."