Pinki Pramanik was remanded in custody for 14 days to await trial on allegations that she repeatedly raped her female live-in partner.
"The court magistrate asked the authorities to constitute a medical board for determining the gender of Pinki," Prabir Roy, who heads the police station on the outskirts of Kolkata where Pramanik was arrested, told AFP.
Pramanik, who retired in 2007, declined to take a medical examination at a government-run hospital when she was questioned on Thursday.
"She has brought false charges against me as I refused to give her 300,000 rupees ($5,400)," Pramanik told reporters from the back of a police van before the court hearing. "Truth will prevail," she said.
Pramanik on Thursday had said she had undergone numerous medical check-ups during her running career and added, "Why should I agree to more ridiculous tests?"
"Pinki poses as a woman but is actually a man," the victim was quoted as saying in Friday’s Calcutta Telegraph. "She would assault me regularly and raped me several times."
She also claimed that Pramanik had promised to marry her.
Pramanik, 26, won gold in the 4x400m relay at the Asian Games and a silver for the same event in the 2006 Commonwealth Games before she stopped competing the following year.
She grew up as the daughter of a poor farmer in rural West Bengal state, about 140 miles (220 kilometres) from Kolkata and has worked as a ticket inspector on the Indian railways.
"My daughter is innocent," Pramanik’s mother, Puspa Pramanik, told a Bengali TV channel. "I don’t believe that she can do such a crime. I hope she will get the right judgement."
Police said Pramanik, who had lived with her partner in Kolkata for several months, will stand trial for rape, criminal assault, cheating and criminal intimidation.
"We don’t know whether it was a case of being male physically or hormonal change over a period of time which can happen," Athletics Federation of India secretary C.K. Valson told the Press Trust of India news agency.
"We have to wait for the medical report and conclusion of the case."
In 2006, Indian athlete Santhi Soundarajan failed a gender test and was stripped of the silver medal she won in the women’s 800m at the Asian Games in Doha.
Santhi insisted along with her parents and coaches that she had done nothing wrong.
Gender controversies are often caused by Congenital Adrenal Hyperplasia (CAH) when females have male physical characteristics or Androgen Insensitivity Syndrome (AIS), when someone is genetically male but their genitals may appear to be female.
South African athlete Caster Semenya won the women’s 800m world championships in 2009 but was then sidelined for 11 months during a probe into her gender.
She was cleared to compete and is a gold medal prospect at the London Olympics.