However, the traffic movement was soon restored, Senior Superintendent of Police Pradip Kumar Yadav said.
The activists, who started the 10-day march from Vrindavan on Friday, were earlier supposed to enter Haryana today.
"After reaching Kosi Kalan town yesterday, the locals along with the villagers decided to block the highway," Sunil Singh, acting president of Yamuna Rakshak Dal that is leading the campaign said.
The activists blocked the Delhi-Agra highway around 9 am today by parking trucks and other vehicles, Yadav said, adding that they later also blocked the Delhi-Agra rail route and stopped goods and passenger trains.
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Over 10,000 marchers, including saints, volunteers, Bharatiya Kisan Union activists and members of religious and social groups, are participating in the campaign.
Their two major demands are that at least 50 per cent of water from Hathini Kund barrage is released into Yamuna and concrete steps are taken to stop the discharge of pollutants, effluents and sewer waste into the river by drains of Delhi.
The protesters are expected to reach Delhi on March 10, where they would hold an indefinite dharna near Parliament.