The 48-year-old was on track to break the record but after eight hours he crashed when he skidded on water he had dropped.
MacDonald, a systems engineer for Cisco who holds the world record for the fastest return cycle ride between Land's End and John O Groats, continued for another five hours but had fallen too far behind the pace.
"Well 20 months of preparation pretty much ended on the back straight after slipping on the water I'd spilled earlier," MacDonald, whose attempt was being masterminded by analysts from Tour de France team Dimension Data, said.
"Did try for the 1,000km record but after the savage pace of the 24 and the injuries it was all too much."
MacDonald, who was planning to ride without toilet breaks at an average of 40kph, eating on the go, rode for 14.5 hours non-stop, covering 516km and 2064 laps of Newport's Geraint Thomas National Velodrome.
"I'd like to thank everyone who made this happen. Too many to list now. Need some rest. Thanks for all the amazing support both in Newport and online, it's been a mad mad day!"
MacDonald had been raising money for African cycling charity Qhubeka, the organisation supported by the Dimension Data Pro Tour team, which provides bicycles for rural communities.
(Reporting by Martyn Herman, editing by Pritha Sarkar)
