Police stepped up security in Lahore and vowed to halt the 1km race.
"We will stop them if they do it," said city police chief Aftab Cheema.
Lahore Mayor Mian Amir has banned races involving women since April, when hard-liners, who regard women's participation in sports as against Islam, attacked competitors at a similar event in the city of Gujranwala.
Critics have voiced concern that men and women will run together and question why the race cannot be held inside a
Asma Jehangir, an activist for the Human Rights Commission of Pakistan - an independent rights group - said on Saturday that the race would begin at 5pm as planned.
"It is our right, and nobody can stop it," Jehangir said.
The informal ban has not been made into law, and it was
unclear how authorities would penalize those who defy it.
The race comes a week after police charged at dozens of
rights activists and detained about 35 of them to prevent
them holding a race in Lahore.
Danyal Ali Hassan, a researcher with New York-based Human Right Watch, and Joseph Francis, the head of the Centre for Legal Aid Assistance and Settlement, were among those arrested. They were freed hours later, but many claimed they had been beaten by police.