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<i>Grace Brown has suffered a puncture during a time trial on stage three of the women's Tour de France as defending champ Demi Vollering took the yellow jersey.</i>

Grace Brown began the third stage of the women's Tour de France on an appropriately gold-coloured bike, but the Olympic time trial champion ended it on her regular Australian TT championship machine after suffering a puncture that killed any hope of victory.

Brown was flying on the the custom-designed bike emblazoned with 'Golden Grace' but instead victory in the short, sharp time trial through the streets of Rotterdam went to defending champion Demi Vollering.

She also took the overall leader's yellow jersey from fellow Dutch rider Charlotte Kool.

Vollering, of the SD Worx-Protime team finished the 6.3km dash over tram rails and bridges in downtown Rotterdam in seven minutes, 25 seconds. Brown was 30th, 24 seconds slower.

American Chloe Dygert, who won the bronze medal at the Paris Olympics individual time trial, and a gold on the Olympic track in the team pursuit, finished second behind Vollering in 7:30.

Vollering now leads the overall standings by three seconds from teammate Lorena Wiebes with Dygert in third. Brown, the best-placed Australian, is 31st, 24 seconds adrift.

With two stages on Tuesday Vollering's victory came hours after Kool beat Wiebes on the line in the race's second stage.

Kool, of the DSM-Firmenich PostNL team passed Olympic road race silver medallist Marianne Vos then overtook Wiebes just before the finishing line in one hour, 32 minutes and 49 seconds. Vos finished third with almost all the peleton given the same time, including Brown.

"Dreams seem to come true quite fast these days. First yesterday with the win and then to do it again with the team today in the yellow jersey, it is just incredible," Kool, who won the opening stage in The Hague on Monday, said.

Stage two had taken the riders 69.7km from Dordrecht to Rotterdam through a typical Dutch landscape of pancake flat polders, waterside dikes and past World Heritage-listed windmills. 

On Wednesday the eight-stage race leaves the southern Dutch town of Valkenburg and heads south into Belgium, finishing in Liege. It then winds through eastern France to finish on Sunday at the top of the punishing climb of the Alpe d'Huez's famous 21 hairpin bends.