The race, organised by a Jewish settler body in the northern West Bank, took place a day after a Palestinian stabbed two Israeli soldiers before being shot dead at a location not far from the finish line.
The route runs from Rosh HaAyin inside Israel then goes eastward through the West Bank to end at the ancient biblical site of Tel Shilo, just outside the Jewish settlement of the same name.
Shahar Tzadok, one of the organisers, told AFP almost all of the runners were Israelis, with only a handful of foreigners taking part.
He was unable to say how many of them were settlers, but religious Jews were also among the runners.
The largely-uphill race began at 5:30 AM local time and passed Ariel, one of the largest settlements in the West Bank, with organisers saying it went off peacefully.
The route is based on a number of verses in the biblical book of 1 Samuel 4 when Israel was defeated by the Philistines in a major battle during which the Ark of the Covenant was seized.
Organisers say the distance was exactly 42 kilometres, describing it as the first-ever marathon.
Israel seized the West Bank along with east Jerusalem and the Gaza Strip during the 1967 Six-Day War and today there are close to 400,000 Israelis living there in more than 130 settlements.
Jewish settlements built on land the Palestinians want for a future state are considered illegal under international law and Israel's ongoing settlement construction has been a major stumbling block to decades of peace efforts.
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