The current study examines the effects of a Tabata high-intensity interval training (HIIT) session on affective, cognitive and physiological indicators in women of different fitness levels. A total of 28 adult women (aged 24.2 ± 1.5 years) completed a 20 m shuttle run test and were then assigned to higher fitness and lower fitness groups (HF and LF, n = 14 each) according to their predicted aerobic power. On a separate occasion, participants completed a 30 min Tabata workout (six 4 min rounds separated by 1 min passive rest). Each round included eight exercises (20 s exercise and 10 s rest). Affective, physiological and cognitive responses were assessed prior to, during and after the protocol. Heart rate and blood lactate concentration increased similarly in both groups over time throughout the workout (p < 0.001). Total Mood Disturbance was higher for LF (111.4 ± 15.7) vs. HF (102.9 ± 11.7) (p = 0.48), vigor showed a level by time interaction of p = 0.006 and Activation–Deactivation Adjective Check List factors deteriorated over time (p < 0.001). The Concentration Grid Test was better overall for HF (10.5 ± 3.6) vs. LF (8.6 ± 3.6) (p = 0.05). The Feeling Scale and Rating of Perceived Exertion worsened similarly in both groups over time (p = 0.002 and p < 0.001, respectively). Positive and negative affect and arousal did not differ between groups or change over time (p > 0.05). These results show that, despite the different levels of aerobic fitness, physiological, metabolic, perceptual and affective responses were similar in the two groups of women during a 30 min Tabata session. This may imply that affective responses during this type of HIIT are independent of aerobic fitness.