Objective To look for cognitive assessment paradigms and electrocardiographic (ECG) characteristics sensitive to mental fatigue through an experimental study. Methods Data were collected from 10 healthy students of the University of Shanghai for Science and Technology who participated in the 4-day National Undergraduate Electronics Design Contest, including their Stanford sleepiness scale (SSS) scores, sleep duration, cognitive task performance, and ECG signal data. Ten ECG features, including time-domain, frequency-domain, and information-domain characteristics, were extracted during cognitive tasks. The cognitive task performance and ECG features sensitive to changes in mental fatigue were analyzed. Results Significant differences were observed in SSS scores (χ²＝23.116, P＜0.001) and sleep duration (χ²＝19.608, P＜0.001) across the 4 d. For cognitive task performance, word-color congruent accuracy and word-color incongruent accuracy in the Stroop task and Single-visual target stimulus accuracy and Visual target-auditory non-target stimulus accuracy in the audiovisual competition task all showed significant negative correlations with SSS scores (all P＜0.05). Regarding ECG features, Poincaré plot SD2 during the Stroop task was positively correlated with sleep duration (P＜0.05), while Poincaré plot SD2 during the 2-back task was positively correlated with mental fatigue assessment scores and was negatively correlated with sleep duration (both P＜0.05). Conclusion The accuracy of the Stroop task and audiovisual competition task is a cognitive ability indicator sensitive to mental fatigue, while the Poincaré plot SD2 during Stroop and 2-back tasks is an ECG indicator sensitive to mental fatigue.