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 used. Subjects competed in male (n = 10; age: 26.3 ± 7.6 years) or mixed-gender (males: n = 4; 28.5 ± 5.7 years; females: n = 6; 28.3 ± 8.1 years) indoor Ultimate Frisbee game play. Games consisted of 10-minute halves, with heart rate (HR), blood lactate concentration ([BLa]), rating of perceived exertion, and 5-m 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 mmol·L vs. 4.68 ± 1.89 mmol·L) than male game play. Significantly (p ≤ 0.05) longer durations were spent at vigorous (male: 60.2 ± 26.1%; mixed-gender: 36.8 ± 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.