Vehicles leaving Gibraltar have since Friday been made to wait nearly six hours to cross the border between Gibraltar and Spain as Spanish authorities searched "practically every vehicle", the government of Gibraltar said.
"The Spanish government has inflicted these unnecessary delays on the elderly, children and the infirm in up to 30 degrees of heat," it added in a statement.
"This torture has resulted in an ambulance being deployed to treat people with medical conditions."
On Friday, a Spanish man caught in the queue to cross the border had to be taken to hospital with chest pains, the statement added.
The Gibraltar authorities were organizing the distribution of bottled water.
Foreign Secretary William Hague telephoned his Spanish counterpart on Sunday to express "serious concerns" over the delays, the Foreign Office said.
In addition to the call, the ministry revealed that Britain had registered a protest with the Spanish ambassador in London.
"Our main concerns at the moment are restoring people's basic right to freedom of movement, and we want to work towards a speedy solution that will help to ease the very difficult situation and humanitarian issues at the border and enable a return to normal operations," said a spokesman.
The row over the border crossing follows earlier accusations of incursions by Spanish fishing boats into Gibraltar's territorial waters.
Gibraltar chief minister Fabian Picardo said he had "absolutely no doubt" that the border traffic delays were linked to a disagreement between Spain and Gibraltar over the placing of concrete blocks to create an artificial reef in Gibraltar's territorial waters.
"This is the way that Spain thinks it is appropriate to retaliate," he told the Gibraltar Broadcasting Corporation.
A contractor hired by the government of Gibraltar placed concrete blocks into the sea around the territory in Spain's southern tip on Wednesday and Thursday to create an artificial reef.
Britain has held Gibraltar since 1713 but Spain wants it returned and refuses to recognise British sovereignty over the waters off the land known as 'the Rock'.
Tiny Gibraltar, just 6.8 square kilometres (2.6 square miles) and home to about 30,000 people, overlooks the only entrance to the Mediterranean Sea from the Atlantic Ocean.