Abstract Background Our previous studies demonstrated that dietary supplementation with milk fat globule membrane (MFGM) combined with habitual exercise improved muscle strength by stimulating neuromuscular development in mice. This study aimed to demonstrate the beneficial effects of dietary MFGM supplementation plus regular exercise on muscle strength and neuromuscular function in healthy humans. Methods The study was designed as a randomized, double-blind, placebo-controlled, crossover trial. Fourteen Japanese adults aged 31–48 years took daily MFGM (1 g) or placebo tablets during the 4-week study period and attended a training program twice a week. Physical function tests and surface electromyography (EMG) were conducted at baseline and at the end of the study period. Results The MFGM group had significantly greater leg extension strength than the placebo group after the 4-week study period. Surface EMG showed that the MFGM group had a significantly higher root mean square amplitude than the placebo group, which indicated that the MFGM group had higher motor unit activity. Conclusions Dietary MFGM supplementation combined with regular exercise improves skeletal muscle strength, which may be due to increased motor unit recruitment in healthy Japanese middle-aged adults.