New measures have been brought in to stop non-essential Tube journeys amid the coronavirus outbreak.
Queues are being introduced at ticket gates and some escalators are being turned off to slow the flow of passengers to platforms.
British Transport Police (BTP) has also deployed 500 officers to patrol the UK's rail network.
London mayor Sadiq Khan said passenger numbers fell by a third on Wednesday morning but more needs to be done.
The Underground network has been hugely reduced by Transport for London (TfL) with 40 stations shut and many services cut back.
Nearly 3,000 people in the capital have tested positive for coronavirus.
"We still need more Londoners to do the right thing and stay at home," said Mr Khan.
"Nearly a third of TfL's staff are now off sick or self-isolating - including train drivers and crucial control centre staff.
"Many of them have years of safety-critical training in order to run specific lines - so it is simply not possible to replace them with others.
"If the number of TfL staff off sick or self-isolating continues to rise - as we sadly expect it will - we will have no choice but to reduce services further."
'Slow the spread'
On Tuesday evening, BTP's assistant chief constable Sean O'Callaghan said officers would be patrolling stations, supporting railway staff and reminding the public of the need to follow the government advice.
"Only those making essential journeys for work should be using the Tube and rail network," he said.
"We strongly urge the rest of the public to do the right thing and help us save lives by staying at home and slowing the spread of the virus."