The results of randomized controlled trials (RCTs) investigating supplemental n-3 polyunsaturated fatty acids (PUFA) on muscle mass and function have been inconsistent. The present study aimed to quantitatively evaluate the effect of n-3 PUFA supplementation on indicators of muscle mass and function in healthy subjects. A systematic literature search was conducted up to July 2020 with databases of PubMed and Web of science. The random-effects model was implemented to calculate the weighted mean difference of net change of indicators regarding muscle mass and function. A total of nine studies (thirteen treatment groups) with 2067 participants were included for data analysis. The summary estimate showed that n-3 PUFA supplementation significantly increased the grip strength (1.17 kg; 95% CI: 0.27, 2.08 kg). Non-significant effect was observed with respect to muscle mass parameters, including fat mass (-0.67 kg; 95% CI: -2.20, 0.87 kg) and lean mass (0.33 kg; 95% CI: -0.35, 1.00 kg). Regarding muscle function indicators, there were non-significant effects on walking speed (-0.01 m•s; 95% CI: -0.03, 0.01 m•s), time up and go test (-0.25 s; 95% CI: -0.55, 0.04 s), respectively. The findings of this study indicated that supplementation with n-3 PUFA might have beneficial effects to improve muscle mass and function in healthy participants. However, there was no significant improvement in the subjects’ muscle mass. Whether n-3 PUFA supplementation has favorable effects in participants with sarcopenia are warranted to be further investigated.