- We investigated effects of irradiance on the relationships between chlorophyll fluorescence transients (OJIP), carotenoid-to-chlorophyll ratio, and fatty acids in a nitrogen-deprived Parietochloris incisa (Chlorophyta, Trebouxiophyceae) Δ5 desaturase mutant accumulating valuable LC-PUFA dihomo-γ-linolenic acid (DGLA). High light (270 μE·m− 2·s− 1 PAR) and nitrogen starvation brought about a decrease in maximum quantum yield of photosystem II (ΦP0ΦP0) and electron transport (ΦE0ΦE0) but enhanced the quantum yield of thermal dissipation (ΦD0ΦD0) and induced non-photochemical quenching (NPQ) in an irradiance-dependent manner. Under high irradiance a decline in the rate of total fatty acid accumulation and DGLA percentage in comparison with the cultures grown under 130 μE·m− 2·s− 1 PAR was recorded. Increasing irradiance from 130 to 270 μE·m− 2·s− 1 enhanced total fatty acid accumulation only within the first week of nitrogen starvation and negatively affected DGLA production. Regardless of irradiance, ΦP0ΦP0, ΦE0ΦE0, and ΦD0ΦD0 exhibited tight (r2 = 0.8–0.9) relationships with the stress-induced changes of total fatty acid and DGLA content and the carotenoid-to-chlorophyll ratio. The applicability and limitations of OJIP and its derived parameters for on-line monitoring of physiological condition and accumulation of value-added products in microalgal cultures grown in photobioreactors are discussed.