The 5-choice serial reaction time task (5-CSRTT) is a valuable cognitive test that permits the simultaneous assessment of several different cognitive modalities, including attention, impulse control, processing speed, and cognitive flexibility. Increasing task difficulty on test days through various challenges can further enhance the versatility of this test by selectively enhancing the cognitive load on different aspects of the task. Systematic comparisons of the effects of different test day challenges on 5-CSRTT performance are essential to verify how these challenges affect different task measures and which manipulations are best suited for future studies of different aspects of cognition. We trained Wistar rats in the 5-CSRTT under standard conditions, then challenged them on the test days by (1) decreasing the duration of the stimulus to be detected, (2) increasing the time interval between trials (intertrial interval, ITI), (3) randomly varying the ITI, or (4) adding a flashing light distractor. All test day challenges produced distinct profiles of performance disruption that reflected differential effects on different cognitive modalities. Decreased stimulus duration selectively impaired attentional performance, while increased ITI increased impulsive-like premature responses and decreased trials completed. Variable ITI induced only mild, nonsignificant disruptions in response inhibition and processing speed, while the flashing light distractor produced comprehensive impairment affecting multiple aspects of 5-CSRTT performance, including disrupted attention and increased premature and timeout responses. This improved understanding of the effects of different test day challenges in the 5-CSRTT will allow researchers to use these manipulations of a valuable cognitive test to their full potential.