Soccer has been categorised as an intermittent sport, where periods of intense exercise are interspersed with periods of active and passive recovery. Performance relies on several factors including the ability to sprint repeatedly, termed as repeated-sprint ability. It is imperative that a scheduled fitness testing programme includes an assessment of this component to ensure maximal performance during match-play. Although there is a plethora of tests available, the aim of this thesis was to develop, validate and evaluate a reactive repeated-sprint test (RRST), and to investigate its use with a variety of age groups and different performance levels. Data was collected during elite soccer matches and high-intensity running distance in the most intense 5- min period was quantified during match-play. The findings led to the development of a soccer-specific RRST, whereby a detailed evaluation was carried out; the validation, reliability and sensitivity was investigated, as well as utilising the RRST to examine maturation trends and correlates with other physical performance tests. The data demonstrate that the RRST is a reliable and valid test that distinguishes between performance across standard, position and seasonal period. It is also suggested that it is sensitive to monitor maturation trends as well as a large correlation with agility. In summary, the present thesis provides new insight into the most intense period and how players can be monitored throughout a season and at different phases of development. It proposes a new fitness test of best practice for soccer clubs; one that can be used as a multi-tool to assess more than one component of fitness (a practical asset due to the busy day-to-day training week of soccer clubs).