Manning, who was born Bradley Manning, stole and leaked a massive cache of military reports and State Department intelligence to WikiLeaks, which has been identified by US intelligence agencies as a front for Russian government operations.
The US Army intelligence specialist has already spent seven years behind bars for handing over classified military and diplomatic files to WikiLeaks in 2010, and another 62 days in jail until last week for refusing to testify before another grand jury.
Representatives for Manning had previously said she was kept in her cell for 22 hours a day, arguing that such solitary confinement threatened her health and amounted to "torture".
Manning has said she objects to grand juries in principle.
US District Judge Anthony Trenga sent her back to jail on Thursday and said that if she did not comply with the subpoena she would after 30 days be fined $US500 ($726) a day.
"I would rather starve to death than to change my opinions in this regard", she said.
Ms. Manning served seven years in a military prison for leaking a trove of documents to WikiLeaks before former president Barack Obama commuted the remainder of her 35-year sentence.
US Attorney Zachary Terwilliger, the top federal prosecutor in the Eastern District of Virginia, told reporters Thursday that his team had "bent over backwards to accommodate" Manning's medical needs and schedule.
Raw video: Former Army intelligence analyst Chelsea Manning says her stance about testifying before a grand jury about her contacts with WikiLeaks has not changed.
"I'm not going to comply with this grand jury", she told journalists outside the Alexandria courthouse before the hearing.
She was released from jail last week after serving a two-month sentence for an earlier refusal to testify.
The fine would go up to $1,000 a day if she continues to refuse to testify after 60 days, the law enforcement official and a spokesman for her attorneys said.
And this grand jury is just coming out of the gate and could be in business for most of the year, which means Manning could be in jail for that long as well.
"I think that this ultimately the goal here is to relitigate the court martial from my perspective".
Manning refused to testify before the grand jury in March, even under an offer of immunity from federal prosecutors.
"The case doesn't make sense, it's very bananas", she said.
"I will not cooperate with this or any other grand jury", Manning insisted.
United States prosecutors are seeking the extradition of Assange from the UK, where he is serving a 50-week jail term for skipping bail, to face charges of conspiracy to commit computer intrusion.
The new grand jury subpoena appears to be related to the USA prosecution of WikiLeaks publisher Julian Assange, who was arrested last month and is now awaiting his extradition hearing in the UK. Manning says she would rather die.