He was freed on bail on Wednesday after spending Easter in jail.
The president has warned the walking campaign is illegal.
The Forum for Democratic Change leader has been charged with unlawful assembly.
Dr Besigye, who was shot in the hand during a similar recent protest, lost to President Yoweri Museveni in a February election he says was rigged.
Before the polls, Dr Besigye had called for Egypt-style uprisings in the event of fraud.
Police responded by banning public demonstrations.
The pair used to be allies, and Dr Besigye was once Mr Museveni's personal physician.