The 70-year-old outspoken billionaire Republican nominee, who has been trailing the Democrat in national polls, questioned the physical stamina of Clinton, 68, arguing that she was more energetic during the beginning of their debate last Sunday, but lost her steam by the end of the debate.
He offered no evidence to back his unsubstatiated claim.
"At the beginning of her last debate, she was all pumped up at the beginning, but at the end she was all 'take me down.' She could barely reach her car," Trump told to loud cheers among his supporters during a rally in New hampshire.
His personal attacks on Clinton have increased lately, with he previously calling her "Crooked Hillary" and that he would send her to jail if he was elected president in the election on November 8.
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He has described Clinton as not having the stamina to be the president of the United States, after the former secretary of state had a scaring ' health episode' when she stumbled and nearly collapsed during the 9/11 memorial event last month.
Clinton was later diagnosed with pneumonia and her health had become a talking point in the electoral race.
Referring to the 17 Republican nominees he has trounced in the primaries, Trump, compared himself with an athlete and said every athlete takes a drug test before the competition.
"We're like athletes, but athletes, they make them take a drug test. We should take a drug test. I think we should take a drug test prior to the debate because I don't know what's going on with her," Trump said.
Trump's accusations against Clinton came as he outlayed his policy to fight the drug abuse in New Hampshire, a state that has become a flashpoint in the battle against the menace.
Clinton was declared a clear winner in first debate. She won the second too according voters in opinion polls, but Trump was declared as having performed "better-than-expected".
Trump has been dwarfed by a barrage of sexual assault allegations, including videos in which he was seen bragging about groping women, and has been virtually abandoned by his own Republican party.
He has been repeatedly cast doubt on the fairness of the US presidential election at election rallies, saying it is being rigged by a corrupt media publishing "outright lies".
"But we are going to stop it. We are not going to back down. We are not going to let it happen," Trump said, pointing out that the latest polls showed he was trailing behind Clinton by a margin greater than the margin of error.
He said the Hillary Clinton documents released by WikiLeaks make clear how much is at stake in this election.
"They show how the Clintons ripped off the people of Haiti. They show the Clinton Campaign viciously attacking Catholics and Evangelicals. They also show how the media collaborates and conspires directly with the Clinton Campaign," he alleged.
Reached by CNN for comment, the Clinton campaign said Trump is trying to depress voter turnout by his "shameful attempts to undermine an election weeks before it happens."
Several of the leading American media houses have endorsed Clinton as president.