Siderophores, small ferric iron (Fe)-binding metabolites, are biomarkers of microbial Fe-stress. Here we report the results from two experiments profiling the distribution of 57Fe-siderophore uptake performed in late summer of 2021 and 2022 near Station ALOHA, the long-term ecological study site of the Hawaii Ocean Time-series program located in the North Pacific Subtropical Gyre. The experiments were designed to investigate how changes in microbial Fe-stress in upper mesopelagic waters (200-400 m) respond to temporal changes in dissolved Fe. Nearly complete uptake (92-99%) of added 57Fe-siderophores was observed in the upper mesopelagic; however, the depths at which siderophore uptake was most active differed between experiments. In 2021 nearly complete uptake of added siderophores occurred at depths between 200 m and 350 m, while in 2022 the region of complete uptake was restricted to waters between 300 m and 350 m. The differences in siderophore uptake between the 200 m and 250 m experiments likely reflects the elevated concentrations of dissolved Fe in the upper mesopelagic waters of 2022, which may have alleviated Fe-stress. Our results confirm the presence of microbial Fe-stress in the upper mesopelagic and suggest that Fe-stress in this region of the water column is responsive to temporal changes in the supply of Fe.