She won an Olympic silver medal in the time trial in 2008 and was world time trial champion in 2010. She has won six UCI Women's Road World Cup one-day races, as well as several stage races including the ten-day Tour de l'Aude. She is three-times a British time trial champion and in 2010 also won the British road race championships.
Pooley retired from professional cycling after the 2014 Commonwealth Games to concentrate on triathlon, duathlon and long-distance running, having won the Lausanne Marathon and the Swissman triathlon the previous year. On 16 December 2015, Pooley announced that she would be returning temporarily to cycling, seeking to qualify to represent Great Britain at the 2016 Summer Olympics in Rio, as she felt the unusually mountainous time trial course on offer played to her strengths
In June 2016, Pooley came out of retirement and re-signed for her former team, Lotto–Soudal Ladies, in time for the 2016 Giro Rosa.
Born in Wandsworth, London, Pooley grew up in Norwich, where she attended Norwich High School for Girls and the sixth form of Norwich School. She began studying for an engineering degree at Trinity Hall, Cambridge in 2001.
She started cycling at university after suffering an injury from cross-country running. At Cambridge she won her university sporting blue for cross-country running and the triathlon.
After a surprise fourth place in the national road championship in 2005, she was signed by UK national team Team Fat Birds UK and rode in the British team supporting Nicole Cooke in the road race at that year's world championships, but crashed out of the race. She rode for the same trade team in 2006 when they were based in Belgium and registered as an international-level UCI Women's Team under the name Team FBUK.
She signed with the Switzerland-based Team Specialized Designs for Women for 2007, with whom she won her first UCI race, stage 3 of the Thüringen Rundfahrt der Frauen, after a 120 km solo escape, the first of many wins from solo breakaways. She represented Britain in the 2007 UCI Road World Championships, finishing 8th in the time trial and 9th in the road race. This earned Great Britain one of their places in the 2008 Summer Olympics.
In 2008, she won the Trofeo Alfredo Binda UCI Road World Cup in Italy after another solo breakaway. She finished 23rd in the Olympic road race on 11 August 2008, where she rode in support of Nicole Cooke's successful gold medal bid but her greatest success of the summer came in the time trial on 13 August, where she won the silver medal behind American Kristin Armstrong.
In 2009, Pooley signed to the Cervélo Test Team, where she remained until their disbandment at the end of 2011. She won the 2009 national time trial championships and took wins for the team in the Coupe du Monde de Montreal, GP de Plouay, and Grand Prix Costa Etrusca one-day races, and the final edition of the Grande Boucle Féminine, once dubbed "the women's Tour de France". Due to financial issues the 2009 Grand Boucle was reduced to only four stages, leading Pooley to joke that it was "more of a Petite Boucle than Grande". She also wore the leader's jersey for three stages of the Giro d'Italia Femminile, but finished the race in fourth place in the general classification after losing the race lead due to her poor descending, which she improved after the 2009 season with the help of her coach Tim Williams and British Cycling psychiatrist Steve Peters.
2010 saw several of Pooley's greatest successes. She won her first major stage race in May, the final edition of the Tour de l'Aude, the longest-running event on the UCI women's calendar. She went on to win another top-level stage race in June, the Giro del Trentino Alto Adige-Südtirol. She also won two UCI Women's Road World Cup one-day races, the La Flèche Wallonne Féminine and the GP de Plouay, and earned a rainbow jersey by winning the time trial at the Road World Championships. She was British national time trial champion for the second year running and also took her only national road race champion's jersey. She finished the year 5th in the 2010 UCI Women's Road World Rankings, the highest end-of-year ranking of her cycling career, and received one of the British Olympic Association's Athlete of the Year trophies, recognising her performance that year as the best by any British cyclist of either sex in any Olympic cycling discipline.
In March 2011, Pooley won the one-day Trofeo Alfredo Binda World Cup race for a second time, again after a lengthy lone breakaway. She broke her collarbone in training on 12 April so was unable to defend her Flèche Wallonne title and did not race again until the women's invitational time trial at the Tour of California on 20 May, in which she placed fifth. Later in the year, however, she won the hilly Tour de l'Ardèche stage race in France and finished second to Marianne Vos in the Giro d'Italia Femminile, both achievements that she would repeat in 2012.
Following the discontinuation of the Garmin Cervélo women's team at the end of 2011, Pooley began riding for the Dutch team AA Drink-Leontien.nl which, in turn, wound up at the end of the 2012 season. At the 2012 Summer Olympics she competed in the Women's road race, helping teammate Lizzie Armitstead to win a silver medal, and placed sixth in the Women's time trial.
Pooley took a step back from pro-racing when she signed for the long-established, but non-UCI registered, Swiss-based Bigla Cycling Team for the 2013 season in order to concentrate on completing her PhD in geotechnical engineering. In a severely reduced season, she missed the 2013 UCI Road World Championships. Pooley managed to win four UCI-ranked races including the six-stage Tour Languedoc Rousillon in May.
Pooley signed for the Lotto Belisol team for 2014. She claimed her third national time trial champion's jersey and won three stages and the mountains classification at the Giro d'Italia Femminile.
Pooley announced during the 2014 Commonwealth Games that she would retire from cycling after competing in the Games' road race in order to concentrate on competing in long distance triathlons and mountain running. Following her retirement announcement, Pooley took the silver medals in both the women's road time trial and road race, and played a key role in helping England teammate Lizzie Armitstead win the road race gold medal.
Pooley made a return to competitive cycling in October 2015 when she competed at the Chrono des Nations time trial, where she finished sixth. In December 2015 she announced that she would aim to compete for Team GB in the 2016 Summer Olympics in Rio de Janeiro after being approached by British Cycling's technical director Shane Sutton, who had studied the Olympic road racing courses and felt that Pooley would have a good chance of winning a medal in the time trial and of helping Lizzie Armitstead to win the road race. She also confirmed that she would continue to compete in triathlon and duathlon competitions in 2016. Pooley made her first appearance in a mass start road race for two years in April 2016 for the British national team at the Women's Tour de Yorkshire, where she helped teammate Alice Barnes to fourth place. In June 2016 Lotto–Soudal Ladies announced that Pooley had rejoined the team for the remainder of the 2016 season, and that she would be part of the team's squad for the 2016 Giro d'Italia Femminile with a role as a domestique for Claudia Lichtenberg. At the Olympics, Pooley rode in the service of Armitstead in the road race, whilst in the time trial she finished 14th, just over two minutes down on winner Kristen Armstrong.
As well as her cycling, she proved herself as a top level marathon runner when she won the Lausanne Marathon in October 2013 with a time of 2:44:29 hours, which placed her inside the top 20 fastest British women in 2013. She has also enjoyed success in triathlon, finishing as top female at the inaugural Swissman triathlon and coming fifth in the Ironman Zurich event. After her retirement from cycling she won the 2014 Powerman Duathlon World Championships in Zofingen, Switzerland at her first attempt, setting a new course record and finishing half an hour ahead of the second-placed finisher.
In February 2015 Pooley won the Challenge Philippines triathlon with a margin of more than ten minutes over the next competitor. She went on to finish ninth at the Ironman Asia-Pacific Championships in Melbourne the following month. Subsequently, in the summer Pooley won the long course race at the Alpe d'Huez Triathlon and the Embrunman triathlon, and successfully defended her Duathlon world title in September.
In March 2016 Pooley took a comfortable win at the Powerman Asia Duathlon Championships in Malaysia, where she finished with a 16-minute lead over the runner up. Following her appearance at the 2016 Olympics, in September she took her third consecutive long-distance duathlon world title with a winning margin of eleven minutes.
Pooley added another international duathlon title when she won the European Powerman Middle Distance Duathlon Championships in Sankt Wendel in May 2017, finishing almost nine minutes ahead of the second-placed finisher.
On 2 January 2015 Pooley was a member of the winning team on Christmas University Challenge, representing Trinity Hall, Cambridge who defeated Balliol College, Oxford, the University of Edinburgh and the University of Hull. Her teammates were international rower Tom James, novelist Adam Mars-Jones and actor Dan Starkey.
Pooley has lived in or near Zurich in Switzerland since 2006, and in December 2013 completed her PhD in geotechnical engineering at ETH Zurich, supervised by Sarah Springman, a former British triathlete who is a vice-president of the International Triathlon Union. She received an honorary Doctorate of Civil Law from the University of East Anglia in July 2012.