The aims of this study were to describe the physiological and fatigue responses associated with indoor Ultimate Frisbee game-play; compare exercise intensities attained to current activity guidelines; and compare responses between male and mixed-gender game formats. A between-subjects (game format) repeated measures (time points) observational experimental design was utilized. Subjects competed in male (n = 10; age: 26.3 ± 7.6 yr) or mixed-gender (males: n = 4; 28.5 ± 5.7 yr; females: n = 6; 28.3 ± 8.1 yr) indoor Ultimate Frisbee game-play. Games consisted of 10-min halves, with heart rate (HR), blood lactate concentration ([BLa-]), rating of perceived exertion, and 5- and 20-m sprint times measured. Durations spent in HR-derived intensity zones and sprint decrements were calculated across games. Mixed-gender game-play produced significantly (P < 0.05) higher relative HR (94.3 ± 5.1 vs.89.6 ± 4.8% HRmax) and [BLa-] (8.31 ± 2.22 vs. 4.68 ± 1.89 mmol•L-1) than male game-play. Significantly (P < 0.05) longer durations were spent at vigorous (male: 60.2 ± 26.1%; mixed-gender36.8 ± 34.8%) and near-maximal (male: 31.6 ± 27.6%; mixed-gender: 58.6 ± 37.7%) exercise intensities than moderate (3.9-7.2%), light (0.7-1.0%), and very light (0-0.1%) intensities in both formats. Limited physiological and sprint fatigue was apparent across games. Subjects primarily performed at vigorous and near-maximal intensities during Ultimate Frisbee. The greater physiological demands encountered during mixed-gender game-play might be attributed to underlying gender-mediated cardiovascular differences. These findings support the efficacy of Ultimate Frisbee as a prescriptive exercise tool for health benefit.