This study investigates the acclimation of N₂-fixing heterocystous cyanobacteria to long-term H₂ photoproduction. Wild-type Calothrix sp. 336/3, Anabaena sp. PCC 7120, and the uptake hydrogenase-deficient mutant (ΔhupL) of Anabaena sp. PCC 7120 were entrapped within Ca²⁺-alginate films and subjected to an argon (Ar) atmosphere containing 6% CO₂. Every third day, the atmosphere was changed to Ar + 6% CO₂ (control), and air or air + 6% CO₂. The air treatments were performed to recover the C / N balance of cells and restore their fitness. After 16–20 h of treatment, the headspace of all vials was again refreshed with Ar + 6% CO₂. Cyanobacteria demonstrated strain-specific differences in carbon allocation and antioxidant responses to different treatments. While glycogen accumulation was observed for both Anabaena strains, Calothrix accumulated significantly less. Instead, Calothrix stored other carbohydrates, likely as extracellular polymeric substances (EPS). All alginate-entrapped cultures demonstrated general increases in oxidative stress over the course of the 450-h experiment. However, specific responses differed, with Calothrix accumulating higher total carotenoid and α-tocopherol levels and demonstrating a more diverse carotenoid profile. This strain also showed a relatively stable D1 protein level across different treatments. In general, all H₂-photoproducing cyanobacteria demonstrated decreases in echinenone content and a shift toward the accumulation of glycosylated carotenoids: myxol 2′-methylpentoside (likely fucoside) in Calothrix and 4-ketomyxol 2′-fucoside in both Anabaena strains. Thus, long-term H₂ photoproduction of immobilized cyanobacteria results in strain-specific acclimation strategies for changing environments.