Timeout expired. The timeout period elapsed prior to completion of the operation or the server is not responding. The statement has been terminated

I have many users on my web site (20000-60000 per day), which is a download site for mobile files. I have remote access to my server (windows server 2008-R2).
I’ve received “Server is unavailable” errors before, but am now seeing a connection timeout error.

I’m not familiar with this – why does it occur and how can I fix it?

The full error is below:

Server Error in “https://stackoverflow.com/” Application. Timeout expired. The timeout period
elapsed prior to completion of the operation or the server is not
responding. The statement has been terminated. Description: An
unhandled exception occurred during the execution of the current web
request. Please review the stack trace for more information about the
error and where it originated in the code.

Exception Details: System.Data.SqlClient.SqlException: Timeout
expired. The timeout period elapsed prior to completion of the
operation or the server is not responding. The statement has been
terminated.

Source Error:

An unhandled exception was generated during the execution of the
current web request. Information regarding the origin and location of
the exception can be identified using the exception stack trace below.

Stack Trace:

[SqlException (0x80131904): Timeout expired. The timeout period
elapsed prior to completion of the operation or the server is not
responding. The statement has been terminated.]
System.Data.SqlClient.SqlConnection.OnError(SqlException exception,
Boolean breakConnection) +404
System.Data.SqlClient.TdsParser.ThrowExceptionAndWarning() +412
System.Data.SqlClient.TdsParser.Run(RunBehavior runBehavior,
SqlCommand cmdHandler, SqlDataReader dataStream,
BulkCopySimpleResultSet bulkCopyHandler, TdsParserStateObject
stateObj) +1363
System.Data.SqlClient.SqlCommand.FinishExecuteReader(SqlDataReader ds,
RunBehavior runBehavior, String resetOptionsString) +6387741
System.Data.SqlClient.SqlCommand.RunExecuteReaderTds(CommandBehavior
cmdBehavior, RunBehavior runBehavior, Boolean returnStream, Boolean
async) +6389442
System.Data.SqlClient.SqlCommand.RunExecuteReader(CommandBehavior
cmdBehavior, RunBehavior runBehavior, Boolean returnStream, String
method, DbAsyncResult result) +538
System.Data.SqlClient.SqlCommand.InternalExecuteNonQuery(DbAsyncResult
result, String methodName, Boolean sendToPipe) +689
System.Data.SqlClient.SqlCommand.ExecuteNonQuery() +327
NovinMedia.Data.DbObject.RunProcedure(String storedProcName,
IDataParameter[] parameters, Int32& rowsAffected) +209
DataLayer.OnlineUsers.Update_SessionEnd_And_Online(Object Session_End,
Boolean Online) +440
NiceFileExplorer.Global.Application_Start(Object sender, EventArgs e)
+163

[HttpException (0x80004005): Timeout expired. The timeout period
elapsed prior to completion of the operation or the server is not
responding. The statement has been terminated.]
System.Web.HttpApplicationFactory.EnsureAppStartCalledForIntegratedMode(HttpContext
context, HttpApplication app) +4052053
System.Web.HttpApplication.RegisterEventSubscriptionsWithIIS(IntPtr
appContext, HttpContext context, MethodInfo[] handlers) +191
System.Web.HttpApplication.InitSpecial(HttpApplicationState state,
MethodInfo[] handlers, IntPtr appContext, HttpContext context) +352
System.Web.HttpApplicationFactory.GetSpecialApplicationInstance(IntPtr
appContext, HttpContext context) +407
System.Web.Hosting.PipelineRuntime.InitializeApplication(IntPtr
appContext) +375

[HttpException (0x80004005): Timeout expired. The timeout period
elapsed prior to completion of the operation or the server is not
responding. The statement has been terminated.]
System.Web.HttpRuntime.FirstRequestInit(HttpContext context) +11686928
System.Web.HttpRuntime.EnsureFirstRequestInit(HttpContext context)
+141 System.Web.HttpRuntime.ProcessRequestNotificationPrivate(IIS7WorkerRequest
wr, HttpContext context) +4863749

EDIT AFTER ANSWERS:
my Application_Start in Global.asax is like below:

protected void Application_Start(object sender, EventArgs e)
{
    Application["OnlineUsers"] = 0;

    OnlineUsers.Update_SessionEnd_And_Online(
        DateTime.Now,
        false);

    AddTask("DoStuff", 10);
}

The stored procedure being called is:

ALTER Procedure [dbo].[sp_OnlineUsers_Update_SessionEnd_And_Online]
    @Session_End datetime,
    @Online bit
As
Begin
    Update OnlineUsers
    SET
        [Session_End] = @Session_End,
        [Online] = @Online

End

I have two methods for getting online users:

1. using Application["OnlineUsers"] = 0;
2. the other one using database

So, for method #2 I reset all OnlineUsers at Application_Start. There are over 482,751 records in that table.