Protein supplementation has shown to improve muscle mass in older adults. However, its effect may be influenced by supplementation dose, frequency and timing. This systematic review aimed to assess the effect of dose, frequency and timing of protein supplementation on muscle mass in older adults. Five databases were systematically searched from inception to 14 March 2023, for randomised controlled trials investigating the effect of protein supplementation on muscle mass in adults aged ≥65 years. Random effects meta-analyses were performed, stratified by population. Subgroups were created for dose (≥30 g, <30 g/day), frequency (once, twice, three times/day) and timing of supplementation (at breakfast, breakfast and lunch, breakfast and dinner, all meals, between meals). Heterogeneity within and between subgroups was assessed using I2 and Cochran Q statistics respectively. Thirty-eight articles were included describing community-dwelling (28 articles, n=3204, 74.6±3.4 years, 62.8 % female), hospitalised (8 articles, n=590, 77.0±3.7 years, 50.3 % female) and institutionalised populations (2 articles, n=156, 85.7±1.2 years, 71.2 % female). Protein supplementation showed a positive effect on muscle mass in community-dwelling older adults (standardised mean difference 0.116; 95 % confidence interval 0.032–0.200 kg, p=0.007, I2=15.3 %) but the effect did not differ between subgroups of dose, frequency and timing (Q=0.056, 0.569 and 3.084 respectively, p>0.05). Data including hospitalised and institutionalised populations were limited. Protein supplementation improves muscle mass in community-dwelling older adults, but its dose, frequency or timing does not significantly influence the effect. •Protein supplementation improves muscle mass in community-dwelling older adults.•This is not associated with the dose, frequency, or timing of supplementation.•Research on protein supplementation and muscle mass gain should target hospitalised and institutionalised populations.