Heat exposure and hypohydration induce physiological and psychological strain during exercise; however, it is unknown if the separate effects of heat exposure and hypohydration are synergistic when co-occurring during loaded exercise. This study compared separate and combined effects of heat exposure and hypohydration on physiological strain, mood state, and visual vigilance during loaded exercise. Twelve males (mean±SD; age, 20±2 years; body mass, 74.0±8.2 kg; maximal oxygen uptake, 57.0±6.0 mLkg-1min-1) completed 4 trials under the following conditions: euhydrated temperate (EUT), hypohydrated temperate (HYT), euhydrated hot (EUH), and hypohydrated hot (HYH). Exercise was 90 min of treadmill walking (∼50% VO2 max, 5% grade) while carrying a 45 lb rucksack. Profile of Mood States and the Scanning Visual Vigilance Test were completed pre and post exercise. The separate effects of heat exposure (EUH) and hypohydration (HYT) on post-exercise Tre were similar (38.25±0.63°C vs. 38.22±0.29°C, respectively, p>0.05), while in combination (HYH), post-exercise Tre was far greater (39.32±0.43°C). Increase in Tre per 1% body mass loss (BML) for HYH (vs. EUH) was greater than HYT (vs. EUT) (0.32°C vs. 0.04°C, respectively, p=0.02); HR increase per 1% BML for HYH (vs. EUH) was 7 bpm compared to HYT (vs. EUT) at 3 bpm (p=0.30). HYH induced greater mood disturbance (post-pre exercise) (35±21 units) compared to other conditions (EUT=3±9 units; HYT=3±16 units; EUH=16±26 units; p<0.001). No differences occurred in visual vigilance (p>0.05). Independently, heat exposure and hypohydration induced similar physiological strain during loaded exercise; when combined, heat exposure with hypohydration, synergistically exacerbated physiological strain and mood disturbance.