Limited research has suggested that acute exposure to negatively charged ions may enhance cardio-respiratory function, aerobic metabolism and recovery following exercise. To test the physiological effects of negatively charged air ions, 14 trained males (age: 32 ± 7 years; V · O 2 max $$ \overset{\cdotp }{V}{\mathrm{O}}_{2 \max } $$ : 57 ± 7 mL min−1 kg−1) were exposed for 20 min to either a high-concentration of air ions (ION: 220 ± 30 × 103 ions cm−3) or normal room conditions (PLA: 0.1 ± 0.06 × 103 ions cm−3) in an ionization chamber in a double-blinded, randomized order, prior to performing: (1) a bout of severe-intensity cycling exercise for determining the time constant of the phase II V · O 2 $$ \overset{\cdotp }{V}{\mathrm{O}}_2 $$ response (τ) and the magnitude of the V · O 2 $$ \overset{\cdotp }{V}{\mathrm{O}}_2 $$ slow component (SC); and (2) a 30-s Wingate test that was preceded by three 30-s Wingate tests to measure plasma [adrenaline] (ADR), [nor-adrenaline] (N-ADR) and blood [lactate] (BLac) over 20 min during recovery in the ionization chamber. There was no difference between ION and PLA for the phase II V · O 2 $$ \overset{\cdotp }{V}{\mathrm{O}}_2 $$ τ (32 ± 14 s vs. 32 ± 14 s; P = 0.7) or V · O 2 $$ \overset{\cdotp }{V}{\mathrm{O}}_2 $$ SC (404 ± 214 mL vs 482 ± 217 mL; P = 0.17). No differences between ION and PLA were observed at any time-point for ADR, N-ADR and BLac as well as on peak and mean power output during the Wingate tests (all P > 0.05). A high-concentration of negatively charged air ions had no effect on aerobic metabolism during severe-intensity exercise or on performance or the recovery of the adrenergic and metabolic responses after repeated-sprint exercise in trained athletes.