The trio of men set the fastest time of 33:44 for their 26.85km lap on the course that will be used for the World Championship road races.
Brown sang the Australia national anthem for the second time in four days. It was her final time trial of her career, with Saturday's road race the final race before retirement.
"We've all won Grand Tour stages and wore Grand Tour leaders jerseys and it was sweet to have a chop with Jay and Michael," O'Connor explained.
Matthews was not sure about it being fun. "I didn't prepare for a time trial, I think I've only ridden my TT bike two or three times since the Tour de France, so it hurt," he explained.
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