Brain-derived neurotrophic factor (BDNF) is a crucial mediator of neuronal plasticity. Here, we investigated the effects of controlled normobaric hypoxia (NH) combined with physical inactivity on BDNF blood levels and executive functions. A total of 25 healthy adults (25.8 ± 3.3 years, 15 female) were analyzed in a randomized controlled cross-over study. Each intervention began with a 30 min resting phase under normoxia (NOR), followed by a 90 min continuation of NOR or NH (peripheral oxygen saturation [SpO2] 85-80%). Serum and plasma samples were collected every 15 min. Heart rate and SpO2 were continuously measured. Before and after each exposure, cognitive tests were performed and after 24 h another follow-up blood sample was taken. NH decreased SpO2 (p < 0.001, ηp2 = 0.747) and increased heart rate (p = 0.006, ηp2 = 0.116) significantly. The 30-min resting phase under NOR led to a significant BDNF reduction in serum (p < 0.001, ηp2 = 0.581) and plasma (p < 0.001, ηp2 = 0.362). Continuation of NOR further significantly reduced BDNF after another 45 min (p = 0.018) in serum and after 30 min (p = 0.040) and 90 min (p = 0.005) in plasma. There was no significant BDNF decline under NH. A 24 h follow-up examination showed a significant decline in serum BDNF, both after NH and NOR. Our results show that NH has the potential to counteract physical inactivity-induced BDNF decline. Therefore, our study emphasizes the need for a physically active lifestyle and its positive effects on BDNF. This study also demonstrates the need for a standardized protocol for future studies to determine BDNF in serum and plasma.