The ad, which is titled "Dangerous
," uses footage of Clinton's fainting episode at a 9/11 anniversary event last month, along with a brief clip of the former secretary of state coughing and a photo of her being assisted up the stairs, to cast doubt on her fitness for office.
"Hillary Clinton doesn't have the fortitude, strength or stamina to lead in our world," the narrator says in the ad, as foreboding music plays in the background.
CNN has reached out to the Clinton campaign for comment and not yet received a response.
The Trump campaign said the ad will "air nationally and will be heavily rotated into battleground states." The campaign has reserved $34.7 million in advertisements between Tuesday and Election Day, according to the ad tracking firm Kantar Media/CMAG.
Questions surrounding Clinton's fitness, though widely debunked, reached a fever pitch within conservative media late this summer, with pro-Trump forces like the Drudge Report and Sean Hannity routinely indulging conspiracy theories that the Democratic nominee is secretly in grave health.
The Trump campaign also gave legitimacy to these theories. In August, Trump spokeswoman Katrina Pierson asserted that Clinton has dysphasia, a brain disorder that affects speech.
The Clinton campaign has condemned those who are pushing the theories, and the candidate's physician has said she is healthy and has not developed any new conditions this year.
The fainting episode last month was the result of pneumonia, for which Clinton had been diagnosed only days earlier.
Trump has repeatedly said that Clinton "doesn't have the stamina" to be president, a charge he repeated at the first presidential debate last month.
But at the end of the second debate on Sunday, Trump offered uncharacteristic praise of his opponent's toughness.
"I will say this about Hillary: She doesn't quit. She doesn't give up. I respect that," Trump said. "I tell it like it is. She's a fighter. I disagree with much of what she's fighting for. I do disagree with her judgment in many cases. But she does fight hard, and she doesn't quit, and she doesn't give up. And I consider that to be a very good trait."