Nearly half of the stations were closed and there was gridlock on the streets of Brazil's largest city, which will host the opening match of the World Cup next Thursday.
Workers are demanding a pay rise of at least 10%.
A new round of negotiations has failed and the strike will continue.
A third of Sao Paulo's metro stations were closed on Friday morning, and there were more than 200km (125 miles) of traffic jams across the city in the morning rush hour.
Police fired tear gas and used batons and shields at a demonstration outside Ana Rosa station in central Sao Paulo.
A military police spokesman said officers intervened after clashes broke out between picketing strikers and commuters trying to enter the station.
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