The aim of this study was to investigate the effect of defense tactical strategy on physiological responses characterizing playing intensity in water-polo game. In the first part of the study, fourteen players were assigned to defending (n = 7) and offending (n = 7) groups and participated in nine 4-min plays applying three different defending systems: press, static-zone and zone-press, in front of the defense court of one goalpost. In the second part, 18 players participated in nine different real full court water-polo games consisting of 3X15min of live-time playing periods. Both in defense court plays and real games, the three defense systems were played in a counterbalanced order and heart rate (HR) was continuously recorded. Additionally, in defense court plays, blood lactate concentration (La) was measured at the end of each 4-min period. Mean HR within defense court plays was higher in press (153 ± 10 beats^.min^-1) than in static-zone (140 ± 11 beats^.min^-1) and zone-press (143 ± 16 beats^.min^-1, p < 0.01). Furthermore, shorter amount of playing time was spent with HR ≤85% of HR peak in press (46.3 ± 22.8%) than in static-zone (81.8 ± 20.5%) and zone-press (75.7 ± 32.0%, p < 0.01). Likewise, mean La was higher in press (6.5±2.9 mmol^.l^-1) than in static-zone (4.7 ± 2.5 mmol^.l^-1) and zone-press (4.6 ± 1.8 mmol^.l^-1, p < 0.01). In real games, however, mean HR was similar between tactical strategies (p > 0.05). Defenders and offenders showed similar HR and La responses across the tactical modes. In conclusion, defense tactical strategies affect physiological responses within a part of the game but do not affect the overall playing intensity of a real water-polo game. Tactical strategies similarly affect offenders and defenders.