Symptoms of chronic obstructive pulmonary disease may vary throughout the day and it is important that therapeutic approaches provide 24-h symptom control. We report the results of two phase IIIb crossover studies, PT003011 and PT003012, investigating the 24-h lung function profile of GFF MDI (glycopyrrolate/formoterol fumarate 18/9.6 μg delivered using innovative co-suspension delivery technology) administered twice daily. Patients with moderate-to-very severe chronic obstructive pulmonary disease received 4 weeks' treatment with each of GFF MDI, placebo MDI, and open-label tiotropium (PT003011 only). Lung function was assessed over 24 h on day 29 of each treatment period. The primary outcome was forced expiratory volume in 1 second area under the curve from 0 to 24 h (FEV AUC ). Other outcomes included change from baseline in average daily rescue medication use over the treatment period. In addition, we conducted a post-hoc analysis of data pooled from both studies to further characterize the effect of GFF MDI on inspiratory capacity. GFF MDI treatment significantly increased FEV AUC versus placebo in studies PT003011 (n = 75) and PT003012 (n = 35) on day 29 (both studies p < 0.0001), with similar improvements in FEV AUC versus placebo for hours 0-12 and 12-24. In PT003011, improvements with GFF MDI versus tiotropium in FEV AUC were greater during hours 12-24 compared to 0-12 h. GFF MDI treatment also resulted in a significant reduction in rescue medication use versus placebo (-0.84 p<0.0001 and -1.11 p=0.0054 puffs/day in PT003011 and PT003012, respectively), and versus tiotropium in PT003011 (-0.44 p=0.017 puffs/day). A post-hoc pooled analysis showed patients treated with GFF MDI were more likely to achieve a >15% increase from baseline in inspiratory capacity than patients treated with placebo or tiotropium (72.1%, 19.0% and 47.0% of patients, respectively after the evening dose on day 29). There were no significant safety/tolerability findings. GFF MDI significantly improved 24-h lung function versus placebo in patients with moderate-to-very severe chronic obstructive pulmonary disease, with similar benefits in the second 12-h period compared to the first, supporting twice-daily dosing of GFF MDI. Pearl Therapeutics, Inc.; www.clinicaltrials.gov ; NCT02347072 and NCT02347085 . Registered 21 January 2015.