While there is evidence that long-chain n-3 PUFA supplementation benefits mood, the extent to which a single high dose of n-3 PUFA can induce acute mood effects has not been examined. The present study investigated whether a single dose of a DHA-rich powder affects self-reported mood in middle-aged males during elevated cognitive demand. In a randomised, double-blind, placebo-controlled trial with a balanced crossover design, twenty-nine healthy males (age M = 52.8 years, sd = 5.3) were administered a powder (in a meal) containing 4·74 g n-3 PUFA (DHA 4020 mg; EPA 720 mg) or placebo in random order on two different testing days separated by a washout period of 7 ± 3 d. Participants completed mood assessments before and after completing two cognitive test batteries at baseline and again 3·5–4·0 h following the consumption of the active treatment or placebo. While completion of the cognitive test batteries increased negative mood, differential effects for alertness (P = 0·008) and stress (P = 0·04) followed consumption of the DHA-rich powder compared with placebo. Although alertness declined when completing the cognitive batteries, it was higher following consumption of the DHA-rich powder compared with placebo (P = 0·006). Conversely, stress was lower following consumption of the DHA-rich powder relative to placebo, though this difference only approached significance (P = 0·05). Overall, results from this pilot study demonstrate that a single high dose of n-3 PUFA may deliver acute mood benefits following elevated cognitive demand in healthy middle-aged males.