Water polo is a team sport characterised by a high intensity, intermittent activity pattern. Intermittent performance tests have been developed and validated to assess match-fitness in several land-based team sports such as soccer, but no such test is available for water polo players. PURPOSE To develop and validate a new intermittent match-fitness test for water polo players. METHODS The Water polo Intermittent Shuttle Test (WIST) consists of repeated 2 ± 7.5 m swims between lane ropes at progressively increasing speeds controlled by audio signals and interspersed by 10 s active recovery. The test finishes when a player can no longer keep up with the required speed. In Phase I of the investigation, 8 male junior players performed the WIST twice, 48 h apart to assess test reliability. To assess test sensitivity and validity, 104 male and female players from different standards of competition and playing positions were tested in Phase II. In Phase III, 16 players performed the WIST five times throughout a competition season to track fitness changes. In Phase IV, 12 players performed the WIST 48 h before four consecutive National League games, and their coaches awarded individual match-fitness scores based on game performances to assess the relationship between match-fitness and test results. Heart rate and blood lactate were measured during and after each test, respectively. RESULTS Phase I: Test-retest values were 216 ± 90 vs 229 ± 96 m (r = 0.98, P < 0.001, CV = 5.4%) for performance, 190 ± 8 vs 192 ± 10 bpm (r = 0.95, P < 0.001, CV = 1.2%) for peak HR, and 7.0 ± 1.8 vs 6.4 ± 1.6 mmol.l−1 (r = 0.84, P < 0.01, CV = 8.8%) for blood lactate. Phase II: Significant differences were observed among different standards of play (ranging from Junior Regional Females 102 ± 10 m to Senior International Males 401 ± 30 m) and playing positions (field players 305 ± 154 m, center forwards 255 ± 118, goal keepers 203 ± 135 m). Phase III: test performance was lower in the early season (344 ± 118 m) than the remainder of the season (range 459 ± 138 to 472 ± 133 m, P < 0.05). Phase IV: WIST performance and match-fitness scores correlated for all field players (r = 0.57, P = 0.05) and also for field players not including center forwards (r = 0.84, P < 0.01). CONCLUSION The WIST is a reliable, sensitive and valid match-fitness test for water polo players. However, test performance of center forwards should not be compared to that of other field players. The WIST could become a useful tool for coaches and sport scientists to assess the effects of different interventions on match-fitness.