In order to explore the effects of constant and altered workload sequences on mental fatigue in a thermoneutral environment, experiments and surveys were carried out in this study. n‐back tasks were used to design different workload sequences. Fifteen healthy right‐handed males were required to experience three different workload sequences for 30 min, respectively, including a constant workload (2‐back task) and 2 altered workload sequences that contained an elevating workload sequence (1‐2‐3‐back tasks) and a reducing workload sequence (3‐2‐1‐back tasks). The PANAS, VAS‐F, and NASA‐TLX scales were selected to investigate changes in the mood, the perceived fatigue, and the perceived workload. Meanwhile, the skin temperature during these three workload sequences was continuously collected. Results from the NASA‐TLX scale indicated that no significant difference in total workload was observed among all three workload sequences. Meanwhile, results from the VAS‐F scale showed that no significant changes in self‐reported mental fatigue were observed among these three workload sequences, which meant that mental fatigue was only related to the total workload. However, self‐reported “energy” from the VAS‐F scale did not reduce significantly during the reducing workload sequence, which meant that the reducing workload sequence could conserve more “energy” than that of the elevating and constant workload sequences. Furthermore, both positive and negative moods changed significantly under the constant workload sequence (2‐back task), but they did not show much changes under altered workloads, which meant that the altered workload sequence could attenuate the mood deterioration. What is more, the mental demand, physical demand, temporal demand, effort, and the total workload increased significantly after both the constant workload and elevating workload sequence, but no significant changes in all these six items of the NASA‐TLX scale were observed under the reducing workload sequence. Finally, the mean skin temperature under the constant workload sequence was lower than that under the altered workload sequences (p > 0.05), but significant changes in skin temperature at the left hand and neck were only observed between the constant and reducing workload sequences. In conclusion, constant and altered workload sequences contributed equally to the mental fatigue in a thermoneutral environment, but the differences in workload sequence produced some differences in mood, energy, and mental demand, which would affect the working performance. Findings of this study provided implications for the proper planning, assignment, and management of tasks in real working settings.