Both Synchronised and According To Pete came to grief, their deaths overshadowing a thrilling victory by 33-1 outsider Neptune Collonges.
The fatalities, following two in last year's event, prompted the British Horseracing Authority to defend safety steps taken in a race which has regularly come under scrutiny for the number of equine casualties it produces.
Since 1973, there have been 33 horse deaths in the showpiece steeplechase, a demanding test of stamina and strength of both horse and jockey.
The sight of 40 horses charging at breakneck speed and thundering over 30 fences makes thrilling viewing. The sight of stricken horses covered by tarpaulins is less palatable.
"We are desperately sad at these two accidents and our sympathies are with the connections of both horses," Aintree racecourse managing director Julian Thick said in a statement.
"When a horse gets hurt, everyone is deeply upset. Safety is the first priority for the organisers of the Grand National and we make every effort to ensure that everyone involved in the event is able to participate in safety.
"Horse racing is a sport that is very carefully regulated and monitored by the BHA, but risk can never be completely removed. The Grand National is a professional and well-organised race and only the best horses and jockeys are allowed to enter."
Following a review of the fatalities in 2011 race some fences were modified to improve safety. Changes included lowering the drop side of the infamous Becher's Brook but this was the fence where Synchronised met his fate on Saturday.
Professor Tim Morris, Director of Equine Science and Welfare for the BHA, said the organisation took the responsibility of looking after the welfare of horse and rider "very seriously".
"It is our stated objective to continue to reduce the number of injuries and fatalities which occur in racing," he said.
Five horses were killed during the four-day Cheltenham Festival meeting last month while the Dubai World Cup was also marred by two deaths in a supporting race at Meydan racecourse in the United Arab Emirates.
Paul Nicholls, the trainer of Neptune Collonges, said all elements of risk in the sport could not be removed.
"All sports have an element of risk and we all take a certain amount of risk each day," he said. "We take every effort to minimise risks but even in Flat races accidents can happen."
Royal Society for the Prevention of Cruelty to Animals equine advisor David Muir said organisers needed to reconsider the number of runners, the number of fences, the length of the race and the type and design of the jumps.
"We've said it before, the death of a horse is the unacceptable face of horse racing. I am not happy with the drop fences and Becher's is a drop fence," he said
"To be fair, the BHA and Aintree management will look into the race and I believe we will see a rolling change without taking away the ethos of the Grand National over the next few years."