The event blending extreme sport — the route is some 160 kilometres long — and historic pilgrimage has drawn participants from around the world.
The runners took off at 6am for the third edition of the "100MeilenBerlin" (100MilesBerlin) in Prenzlauer Berg, which since Germany's 1990 reunification has been transformed from an East Berlin working-class district into a fashionable neighbourhood.
They will pass through a rotunda near the famous border crossing Checkpoint Charlie, which currently houses a large panorama installation by artist Yadegar Asisi depicting daily life along the Wall in the 1980s.
Designed to stop an exodus of people to the West, it cut through city blocks, neighbourhoods, fields and forests and was shadowed by a heavily mined "death strip", overlooked by watch towers and patrolled by soldiers and dogs.
By remembering Germany's painful division, "we can all help to ensure that this doesn't happen again, at least in Germany," said the oldest registered participant, Sigrid Eichner, 73, who also lamented the continuing division of North and South Korea.
The Berlin woman, who has lived in the city's east since 1974, is a legendary veteran of more than 1,800 running races around the world equal to or longer than the classic marathon format.
These days, the path of the Wall is a hiking and cycling trail, which takes a seasoned ultramarathon runner 16 to 30 hours to complete, depending in part on the weather. Forecasts for Saturday's event was for summer showers and temperatures up to 20 degrees Celsius (68 degrees Fahrenheit).
Promoted as a marriage of "sport and history", the ultramarathon, like previous editions in 2011 and 2013, was dedicated to one of the "victims of the Wall" who paid with their lives trying to escape.
Each runner who makes the full distance will receive a medal with the image of Peter Fechter, who was 18 years old when he was shot dead by border guards during a 1962 escape attempt near Checkpoint Charlie.