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The fund is down 15.9% through October 27, according to a performance update. The fund had been down 11.2% a week ago.
The fund's performance was primarily dragged down by its largest equity holding, Valeant Pharmaceuticals.
Since taking a position earlier this year, Pershing Square has likely lost more than $1 billion on paper on its investment.
Shares of the Canadian drug company have collapsed since last Wednesday after the California-based short-selling firm Citron Research published a report asking whether Valeant was operating an Enron-like fraud.
Valeant collapse
Valeant's share price has declined by more than 34% since the report came out.
Valeant has categorically denied the allegations in the Citron report. The company hosted an all-hands call Monday morning to address the allegations.
Ackman's Pershing Square Capital Management owns 21,473,933 shares of Valeant, including 2 million shares Ackman bought during last Wednesday's sell-off. Pershing Square is Valeant's third-largest shareholder.
Ackman first disclosed his position in Valeant March 17, when the share price was $200 per share. At that share price, the position was worth $3.9 billion.
With his added shares from last week, Ackman's position is worth about $2.36 billion at Tuesday's closing share price of $109.54. That would represent a loss of around $1.5 billion.
Pershing Square was the best-performing hedge fund last year, returning 40.4%.
Ackman plans to hold a conference call Friday, the last trading day of the month, to discuss his Valeant investment.
Meanwhile, a number of other positions in his portfolio, including Platform Specialty Products and Canadian Pacific, have declined this year. His short of Herbalife has continued to rise during 2015.