Acute bouts of uninterrupted sitting has been associated with discomfort and fatigue in adult populations. However, little is known regarding the impact of uninterrupted sitting on such outcomes among college students. Understanding these relations would be useful for informing best practice and future interventions. The present study explored the relation between uninterrupted sitting and perceived levels of physical discomfort and sleepiness among college students in a real classroom setting. We recruited 54 undergraduate students enrolled in a single class at a Midwestern university. Participants remained seated throughout a 2.5 h lecture while completing the Stanford Sleepiness Scale (SSS) and General Comfort Scale (GCS) every 15 min. Linear mixed effect model analyses were used to determine the relations between the independent and dependent variables and the duration at which students reported significant impairments in discomfort and/or sleepiness. Classroom sitting time was associated with increases in discomfort (r = 0.28, p < 0.01) and sleepiness (r = 0.30, p < 0.01). Students reported significant impairments in discomfort and sleepiness after 75 and 15 min, respectively. These findings support further research into the acceptability, feasibility and efficacy of interventions designed to interrupt classroom sitting on discomfort, sleepiness and measures of academic performance.