The southeastern state of Minas Gerais was the worst hit, with some 14 highways blocked off, causing bottlenecks on motorways leading to the business hubs of Sao Paulo and Belo Horizonte.
Protesters set fire to a bus and vandalized three others on a highway linking Belo Horizonte with the capital Brasilia, bringing traffic to a halt for some six hours, O Estado de Sao Paulo daily reported.
Following a decision by the authorities to impose fines on those joining the blockade, driver union MUBC told truckers to move off early Wednesday - but at a snail's pace.
The protesters had blocked off the main access road to Latin America's biggest container port at Santos in the southeast for 24 hours before it re-opened late Tuesday.
The protests come amid the worst social unrest to hit Brazil in two decades, with hundreds of thousands having taken to the streets in recent weeks to rally against poor public services and rampant corruption.
The unrest began three weeks ago and continued through the Confederations Cup, a dress rehearsal for next year's World Cup.
The protesters have objected to the $15 billion price tag for the sports jamborees, saying much of it is has been misspent and should have instead been invested in infrastructure and social projects.
Protesters had more cause for complaint Wednesday when it emerged that Henrique Eduardo Alves, chairman of the country's chamber of lawmakers, had flown on a military plane to Rio de Janeiro to see the hosts beat Spain in Sunday's Confederations Cup final.
After Folha de Sao Paulo newspaper published the story, Alves's office told AFP he would be paying back the cost of the trip.
President Dilma Rousseff responded to the unrest by submitting a proposal to Congress on Tuesday to hold a referendum on political reform.
On Wednesday, the Brazilian senate approved an amended bill to divert profits from the country's lucrative oil sector toward investment in education and health.
The move would see 75 percent of oil sector profits assigned to education and the remainder to health, the official senate bulletin said.
An earlier government proposal to pour all oil profits into education had long been stalled, but the protests of recent weeks galvanized politicians into action 16 months ahead of presidential elections.
The lower house of Congress approved the proposal last week but will have to do so again after the Senate made amendments, including assigning some cash to a creaking public health sector.
Another amendment provides for 50 percent of eventual receipts from deep water pre-salt deposits under exploration to go directly to education, with the remainder split between both education and health.
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