We aimed to investigate the long-term effect of daily Calanus oil supplementation on maximal oxygen uptake (VO2max) in healthy 30- to 50-year-old participants. The study was motivated by preclinical studies reporting increased VO2max and metabolic health with omega-3 rich Calanus oil. In a double-blinded study, 71 participants were randomized to receive 2 g/day of Calanus or placebo supplementation for a total of 6 months. The participants underwent exercise testing and clinical investigations at baseline, 3 months, and 6 months. Main study endpoint was change in VO2max from baseline to 6 months. Fifty-eight participants completed the 6-month test and were included in the final data analysis (age: Calanus, 39.7 [38.0, 41.4] and placebo, 38.8 [36.8, 40.9] years; body mass index: Calanus, 24.8 [24.0, 25.6] and placebo, 24.8 [23.7, 25.8] kg/m2; and VO2max: Calanus, 50.4 [47.1, 53.8] and placebo, 50.2 [47.2, 53.1] ml·kg−1·min−1). There were no between-group differences at baseline, nor were there any between-group differences in absolute (Calanus, 3.74 [3.44, 4.04] and placebo, 3.79 [3.44, 4.14] L/min) or relative VO2max (Calanus, 49.7 [46.2, 53.2] and placebo, 49.5 [46.0, 53.1] ml·kg−1·min−1) at 6 months (mean [95% confidence interval]). There were no between-groups change in clinical measures from baseline to 3 and 6 months. In conclusion, VO2max was unaffected by 6 months of daily Calanus oil supplementation in healthy, physically fit, normal to overweight men and women between 30 and 50 years old.